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Abstract:

The present disclosure provides a novel technology that involves improved
primer design. These primer pairs have a wide range of applications and
provide high sensitivity and specificity.

Claims:

1. (canceled)

2. A polynucleotide primer combination comprising a first polynucleotide
(1) and a second polynucleotide (2), the first polynucleotide (1)
comprising a first domain (a) having a sequence that is sufficiently
complementary to a first target polynucleotide region (A), a second
domain (c) comprising a unique polynucleotide sequence, and a third
domain (b) comprising a polymer sequence that is not sufficiently
complementary to hybridize to a domain in the first polynucleotide (1), a
domain in the second polynucleotide (2), or a domain in the target
polynucleotide, wherein domains in the first polynucleotide are arranged
5'-a-b-c-3'; the second polynucleotide (2) comprising a first domain (f)
having a sequence that is sufficiently complementary to a sequence in a
second target polynucleotide region (F), a second domain (d) comprising a
polynucleotide sequence sufficiently complementary to (c) such that (c)
and (d) will hybridize under appropriate conditions, and a third domain
(e) comprising a polynucleotide sequence that is not sufficiently
complementary to hybridize to a domain in the first polynucleotide (1), a
domain in the second polynucleotide (2), or a domain in the target
polynucleotide, wherein domains in the second polymer are arranged
5'-d-e-f-3', wherein under conditions in which region (A) specifically
hybridizes to domain (a) and region (F) specifically hybridizes to domain
(f), domain (c) hybridizes to domain (d) and neither domain (b) nor
domain (e) hybridizes to a domain in the first polynucleotide (1), a
domain in second polynucleotide (2) or a domain in the target
polynucleotide.

3-6. (canceled)

7. The polynucleotide primer combination of claim 2, wherein the second
polynucleotide further comprises a fourth domain (h) positioned between
domain (f) and domain (e), wherein domain (h) comprises a replication
blocker and wherein domain (h) is not part of domain (f).

8. The polynucleotide primer combination of claim 7, wherein domain (h)
is part of domain (e).

9. The polynucleotide primer combination of claim 8, wherein domain (h)
is at a position in domain (e) that is 1, 2, 3, 4 or 5 nucleotides from
the 3' end of domain (e).

10-38. (canceled)

39. The polynucleotide primer combination of claim 2 wherein the sequence
of domain (f) is 100% complementary to the sequence of the second
non-target polynucleotide region (F*).

40. (canceled)

41. The polynucleotide primer combination of claim 2 wherein the sequence
of domain (f) comprises a mismatch with the sequence of the non-target
polynucleotide region (F*).

42. The polynucleotide primer combination of claim 41 wherein the
mismatch in the sequence of domain (f) with respect to the sequence of
the non-target polynucleotide region (F*) is not a 3' base mismatch.

43-45. (canceled)

46. The polynucleotide primer combination of claim 2 further comprising a
blocker polynucleotide that comprises a 5' terminus and a 3' terminus.

47-55. (canceled)

56. The polynucleotide primer combination of claim 46 wherein the blocker
polynucleotide is sufficiently complementary to hybridize under
appropriate conditions to a third target polynucleotide region (X),
wherein region (X) is between region (A) and region (F).

57. The polynucleotide primer combination of claim 56 wherein region (X)
comprises a sequence that is at least about 1 nucleotide to about 100
kilobases in length.

58. The polynucleotide primer combination of claim 57 further comprising
a first rigid frame blocker polynucleotide (RF1) and a second rigid frame
blocker polynucleotide (RF2).

59. The polynucleotide primer combination of claim 58 wherein
polynucleotide (RF1) comprises a domain (q) that is sufficiently
complementary to a region (Xq) in a non-target polynucleotide to allow
hybridization between domain (q) and region (Xq) under appropriate
conditions, a domain (t) that is sufficiently complementary to a region
(Xt) in the non-target polynucleotide to allow hybridization to allow
hybridization between domain (t) and region (Xt), and a domain (r) that
is sufficiently complementary to a domain (v) in polynucleotide (RF2) to
allow hybridization between domain (r) and domain (v) under appropriate
conditions; and wherein polynucleotide (RF2) comprises a domain (u) that
is sufficiently complementary to a region (Xu) in a non-target
polynucleotide to allow hybridization between domain (u) and region (Xu)
under appropriate conditions, a domain (w) that is sufficiently
complementary to a region (Xw) in the non-target polynucleotide to allow
hybridization between domain (w) and region (Xw), and domain (v) that is
sufficiently complementary to domain (r) in polynucleotide (RF1) to allow
hybridization between domain (r) and domain (v) under appropriate
conditions, and wherein when domain (q) is specifically hybridized to
region (Xq), and domain (t) is specifically hybridized to region (Xt),
and domain (u) is specifically hybridized to region (Xu), and domain (w)
is specifically hybridized to region (Xw), and domain (r) is specifically
hybridized to domain (v), and domain (a) is specifically hybridized to
region (A), domain (f) will not hybridize to region (F), and wherein
region (Xq), region (Xt), region (Xu), region (Xu) and region (Xw) are
not in the target polynucleotide.

60-66. (canceled)

67. The polynucleotide primer combination of claim 2 wherein region (A)
is 5' to region (F) in the target polynucleotide.

68. The polynucleotide primer combination of claim 67 wherein at least
one nucleotide in domain (a) overlaps at least one nucleotide in domain
(f).

69-73. (canceled)

74. A method of detecting a target polynucleotide in a sample with a
primer combination, the primer combination comprising a first
polynucleotide (1) and a second polynucleotide (2), the first
polynucleotide (1) comprising a first domain (a) having a sequence that
is sufficiently complementary to a first target polynucleotide region
(A), a second domain (c) comprising a unique polynucleotide sequence, and
a third domain (b) comprising a sequence that is not sufficiently
complementary to hybridize to a domain in the first polynucleotide (1), a
domain in the second polynucleotide (2), or a domain in the target
polynucleotide, or the third domain comprises a chemical polymer, the
second polynucleotide (2) comprising a first domain (f) that is fully
complementary to a second target polynucleotide region (F), a second
domain (d) comprising a polynucleotide sequence sufficiently
complementary to domain (c) such that domain (c) and domain (d) will
hybridize under appropriate conditions, and a third domain (e) comprising
a sequence that is not sufficiently complementary to hybridize to a
domain in the first polynucleotide (1), a domain in the second
polynucleotide (2), or a domain in the target polynucleotide, domain (f)
having a sequence that is not fully complementary to a non-target
polynucleotide in the sample and the method comprising the steps of:
contacting the sample with the primer combination and a polymerase under
conditions that allow extension of a sequence from domain (f) which is
complementary to the target polynucleotide when the second target
polynucleotide region (F) is present in the sample and detecting the
sequence extended from domain (f) indicating the second target
polynucleotide region (F) is present in the sample.

75. (canceled)

76. A method of detecting a target polynucleotide in a sample with a
primer combination of claim 2 wherein the second polynucleotide (2)
comprises a first domain that is fully complementary to region (F) and
wherein domain (f) is not fully complementary to a non-target
polynucleotide region in the sample, the method comprising the steps of:
contacting the sample with the primer combination and a polymerase under
conditions that allow extension of a sequence from domain (f) which is
complementary to the target polynucleotide when the second target
polynucleotide region (F) is present in the sample and detecting the
sequence extended from domain (f).

77-81. (canceled)

82. A method of initiating polymerase extension using a primer
combination and a target polynucleotide as template in a sample, the
primer combination comprising a first polynucleotide (1) and a second
polynucleotide (2), the first polynucleotide (1) comprising a first
domain (a) having a sequence that is sufficiently complementary to a
first target polynucleotide region (A), a second domain (c) comprising a
unique polynucleotide sequence, and a third domain (b) comprising a
sequence that is not sufficiently complementary to hybridize to a domain
in the first polynucleotide (1), a domain in the second polynucleotide
(2), or a domain in the target polynucleotide, or the third domain
comprises a chemical polymer, the second polynucleotide (2) comprising a
first domain (f) that is fully complementary to a second target
polynucleotide region (F), a second domain (d) comprising a
polynucleotide sequence sufficiently complementary to domain (c) such
that domain (c) and domain (d) will hybridize under appropriate
conditions, and a third domain (e) comprising a sequence that is not
sufficiently complementary to hybridize to a domain in the first
polynucleotide (1), a domain in the second polynucleotide (2), or a
domain in the target polynucleotide, domain (f) having a sequence that is
not fully complementary to a non-target polynucleotide in the sample, and
wherein the sample comprises a mixture of (i) a target polynucleotide
that has a sequence (F) that is fully complementary to the sequence in
domain (f) and (ii) a non-target polynucleotide that has a sequence (F*)
that is not fully complementary to (f), wherein the sequence of (F) is
identical to the sequence of (F*) except for at least a one nucleotide
difference, the method comprising the step of contacting the sample with
the primer combination and a polymerase under conditions that allow
extension of a sequence from domain (f) and complementary to the target
polynucleotide strand when domain (f) contacts region (F).

83-86. (canceled)

87. A method of initiating polymerase extension using the primer
combination of claim 2 and a target polynucleotide as template in a
sample, wherein the second polynucleotide (2) comprises a first domain
(f) that is fully complementary to a first target polynucleotide region
(F) and wherein domain (f) is not fully complementary to a non-target
polynucleotide in the sample, the method comprising the steps of:
contacting the sample with the primer combination and a polymerase under
conditions that allow extension of a sequence from domain (f) which is
complementary to the target polynucleotide when the target polynucleotide
is present in the sample.

88-92. (canceled)

93. A method of amplifying a target polynucleotide in a sample using a
polynucleotide primer combination, the primer combination comprising a
first polynucleotide (1) and a second polynucleotide (2), the first
polynucleotide (1) comprising a first domain (a) having a sequence that
is sufficiently complementary to a first target polynucleotide region
(A), a second domain (c) comprising a unique polynucleotide sequence, and
a third domain (b) comprising a sequence that is not sufficiently
complementary to hybridize to a domain in the first polynucleotide (1), a
domain in the second polynucleotide (2), or a domain in the target
polynucleotide, or the third domain comprises a chemical polymer, the
second polynucleotide (2) comprising a first domain (f) that is fully
complementary to a second target polynucleotide region (F), a second
domain (d) comprising a polynucleotide sequence sufficiently
complementary to domain (c) such that domain (c) and domain (d) will
hybridize under appropriate conditions, and a third domain (e) comprising
a sequence that is not sufficiently complementary to hybridize to a
domain in the first polynucleotide (1), a domain in the second
polynucleotide (2), or a domain in the target polynucleotide, domain (f)
having a sequence that is not fully complementary to a non-target
polynucleotide in the sample and wherein the sample comprises a mixture
of (i) a target polynucleotide that has a sequence in region (F) that is
fully complementary to the sequence in domain (f) and (ii) one or more
non-target polynucleotides that are not fully complementary to domain
(f); the method comprising the steps of: (a) contacting the sample with
the primer combination and a polymerase under conditions that allow
extension of a sequence from domain (f) which is complementary to the
target polynucleotide when the target polynucleotide is present in the
sample, (b) denaturing the sequence extended from domain (f) from the
target polynucleotide, and (c) repeating step (a) in the presence of a
reverse primer having a sequence complementary to a region in the
sequence extended from domain (f) in step (b) to amplify the target
polynucleotide, wherein extension and amplification of the target
polynucleotide occurs when region (F) is fully complementary to the
sequence in the domain (f) but is less efficient or does not occur when
region (F) in the target polynucleotide is not fully complementary to the
sequence in domain (f).

94. A method of amplifying a target polynucleotide in a sample using a
polynucleotide primer combination of claim 2, wherein the second
polynucleotide (2) comprises a first domain (f) that is fully
complementary to a first target polynucleotide region (F) and wherein
domain (f) is not fully complementary to a non-target polynucleotide in
the sample, the method comprising the steps of: (a) contacting the sample
with the primer combination and a polymerase under conditions that allow
extension of a sequence from domain (f) which is complementary to the
target polynucleotide when the target polynucleotide is present in the
sample, (b) denaturing the sequence extended from domain (f) from the
target polynucleotide, and (c) repeating step (a) in the presence of a
reverse primer having a sequence complementary to a region in the
sequence extended from (f) in step (b) to amplify the target
polynucleotide, wherein extension and amplification of the target
polynucleotide occurs when region (F) is fully complementary to the
sequence in domain (f) but is less efficient or does not occur when the
first region in the target polynucleotide is not fully complementary to
the sequence in domain (f).

95-101. (canceled)

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit under 35 U.S.C.
§119(e) of U.S. Provisional Application No. 61/479,344, filed Apr.
26, 2011, which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The disclosure relates to polynucleotide combinations and their
use.

SEQUENCE LISTING

[0003] This application contains, as a separate part of the disclosure, a
Sequence Listing in computer-readable form (filename:
46157A_SeqListing.txt; created: Apr. 25, 2012; 74,958 bytes--ASCII text
file) which is incorporated by reference in its entirety.

BACKGROUND

[0004] Detection and amplification of nucleic acids play important roles
in genetic analysis, molecular diagnostics, and drug discovery. Many such
applications require specific, sensitive and inexpensive quantitative
detection of certain DNA or RNA molecules, gene expression, DNA mutations
or DNA methylation present in a small fraction of total polynucleotides.
Many current methods use polymerase chain reaction, or PCR, and
specifically, real-time PCR (quantitative, or qPCR) to detect and
quantify very small amounts of DNA or RNA from clinical samples.

[0005] While the performance of current PCR assays is constantly
improving, their sensitivity, specificity and cost are still far away
from becoming a widely acceptable diagnostic test. Indeed, many PCR
methods currently used in the art suffer from technical limitations that
make the methods inadequate for many practical applications. For example,
in instances where the target molecule has secondary structure that
inhibits or even completely prevents binding of one or both primers to
the target, amplification can be reduced or even non-existent, which, for
example, from a diagnostic standpoint could give rise to a false negative
despite use of a highly specific primer with binding properties that
would be expected to be sensitive. Other challenges include low
sensitivity of current real-time PCR assays in detection and
discrimination of rare DNA molecules with a single base mutation in
situations when they mixed with thousands of non-mutated DNA molecules,
and ability to combine multiple mutation detection assays into one
multiplex diagnostic assay.

[0006] There thus remains a need in the art for a development of
amplification primers that combines high binding specificity with low
synthesis cost that retain the ability to overcome technical problems
recognized in the art, including novel application of PCR for diagnostics
using next generation sequencing platforms.

SUMMARY OF THE INVENTION

[0007] In one aspect, the disclosure provides a polynucleotide primer
combination comprising a first polynucleotide (1) and a second
polynucleotide (2), the first polynucleotide (1) comprising a first
domain (a) having a sequence that is sufficiently complementary to a
first target polynucleotide region (A), a second domain (c) comprising a
unique polynucleotide sequence, and optionally a third domain (b)
comprising a polymer sequence that is not sufficiently complementary to
hybridize to a domain in the first polynucleotide (1), a domain in the
second polynucleotide (2), or a region in the target polynucleotide,
wherein domains in the first polynucleotide are arranged 5'-a-b-c-3'; the
second polynucleotide (2) comprising a first domain (f) having a sequence
that is sufficiently complementary to a sequence in a second target
polynucleotide region (F), a second domain (d) comprising a
polynucleotide sequence sufficiently complementary to (c) such that (c)
and (d) will hybridize under appropriate conditions, and a third domain
(e) comprising a polynucleotide sequence that is not sufficiently
complementary to hybridize to a domain in the first polynucleotide (1), a
domain in the second polynucleotide (2), or a region in the target
polynucleotide, wherein domains in the second polymer are arranged
5'-d-e-f-3', wherein under conditions in which region (A) specifically
hybridizes to domain (a) and region (F) specifically hybridizes to domain
(f), domain (c) hybridizes to domain (d) and neither domain (b) nor
domain (e) hybridizes to a domain in the first polynucleotide (1), a
domain in second polynucleotide (2) or a region in the target
polynucleotide.

[0008] In another embodiment, the disclosure provides a polynucleotide
primer combination comprising a first polynucleotide (1) and a second
polynucleotide (2), the first polynucleotide (1) comprising a first
domain (a) having a sequence that is sufficiently complementary to a
first target polynucleotide region (A), a second domain (c) comprising a
unique polynucleotide sequence, and a third domain (b) comprising a
polymer sequence that is not sufficiently complementary to hybridize to a
domain in the first polynucleotide (1), a domain in the second
polynucleotide (2), or a domain in the target polynucleotide, wherein
domains in the first polynucleotide are arranged 5'-a-b-c-3'; the second
polynucleotide (2) comprising a first domain (f) having a sequence that
is sufficiently complementary to a sequence in a second target
polynucleotide region (F), a second domain (d) comprising a
polynucleotide sequence sufficiently complementary to (c) such that (c)
and (d) will hybridize under appropriate conditions, and optionally a
third domain (e) comprising a polynucleotide sequence that is not
sufficiently complementary to hybridize to a domain in the first
polynucleotide (1), a domain in the second polynucleotide (2), or a
domain in the target polynucleotide, wherein domains in the second
polymer are arranged 5'-d-e-f-3', wherein under conditions in which
region (A) specifically hybridizes to domain (a) and region (F)
specifically hybridizes to domain (f), domain (c) hybridizes to domain
(d) and neither domain (b) nor domain (e) hybridizes to a domain in the
first polynucleotide (1), a domain in second polynucleotide (2) or a
domain in the target polynucleotide.

[0009] In some embodiments, polynucleotide primer combinations are
provided wherein domain (c) is at least 70% complementary to domain (d),
and in some aspects polynucleotide primer combinations are provided
wherein domain (d) is at least 70% complementary to domain (c). In
further aspects, domain (c) is at least 75%, at least 80%, at least 85%,
at least 90%, at least 95% or more complementary to domain (d).
Embodiments are also provided in which domain (d) is at least 75%, at
least 80%, at least 85%, at least 90%, at least 95% or more complementary
to domain (c). The disclosure also provides embodiments wherein domain
(d) and domain (c) are sufficiently complementary to hybridize to each
other in the absence of the template polynucleotide.

[0010] In various aspects, the polymer sequence of domain (b) is selected
from the group consisting of a polynucleotide sequence, a polynucleotide
sequence comprising at least one modified nucleotide, and a
non-polynucleotide chemical polymer sequence. In one embodiment, the
polymer sequence of domain (b) is a chemical polymer sequence that is
hydrophilic.

[0011] In further embodiments, the polynucleotide sequence of domain (e)
is selected from the group consisting of a polynucleotide sequence of
naturally-occurring nucleotides and a polynucleotide sequence comprising
at least one modified nucleotide, wherein the polynucleotide sequence of
domain (e) is a template for polymerase extension.

[0012] Polynucleotide primer combinations of the disclosure include, in
some aspects, those that further comprise a domain (g) positioned between
domain (d) and domain (e) in the second polynucleotide, wherein domain
(g) comprises a replication blocker. In still further aspects, the second
polynucleotide further comprises a fourth domain (h) positioned between
domain (f) and domain (e), wherein domain (h) comprises a replication
blocker and wherein domain (h) is not part of domain (f). In one aspect,
domain (h) is part of domain (e) while in various aspects, domain (h) is
at a position in domain (e) that is 1, 2, 3, 4, or 5 nucleotides from the
3' end of domain (e). Any moiety that can block extension of a
polynucleotide by a polymerase enzyme is contemplated, and in certain
aspects the replication blocker is selected from the group consisting of
a modified base, an abasic site and a polymer. In some embodiments, the
second polynucleotide (2) comprises both a domain (g) and a domain (h).

[0013] The disclosure also provides polynucleotide primer combinations in
which the second polynucleotide (2) further comprises a domain (s)
positioned 5' of domain (d), wherein domain (s) comprises a sequence that
is not sufficiently complementary to the first polynucleotide (1) or the
second polynucleotide (2) to hybridize to the first polynucleotide (1) or
the second polynucleotide (2) under conditions in which domain (a)
specifically hybridizes to region (A), and optionally wherein domain (s)
is sufficiently complementary to a polynucleotide extended from domain
(f) to hybridize to the polynucleotide extended from domain (f) under
conditions in which domain (a) specifically hybridizes to region (A). In
some embodiments, domain (s) further comprises a detectable marker, and
in various aspects domain (s) further comprises a quencher that quenches
the detectable marker.

[0014] In some aspects, polynucleotide primer combinations are provided
wherein domain (a) of the first polynucleotide (1) further comprises a
domain (j), wherein domain (j) is contiguous with domain (a) and is
positioned 3' of domain (a), and wherein domain (j) is sufficiently
complementary to a region (F*) in a non-target polynucleotide to
hybridize under conditions in which domain (a) specifically hybridizes to
region (A), wherein (i) region (F*) in the non-target polynucleotide
differs from region (F) in the target polynucleotide at at least one
nucleotide or (ii) region (F*) comprises a sequence that is identical to
region (F).

[0015] In further aspects, domain (a) and domain (j) are not contiguous
and are separated by a domain (p), wherein domain (p) is not sufficiently
complementary to a domain in the first polynucleotide (1), a domain in
the second polynucleotide (2), or the target polynucleotide to hybridize
to the first polynucleotide (1), the second polynucleotide (2) or the
target polynucleotide under conditions wherein domain (a) specifically
hybridizes to region (A).

[0016] In any of the polynucleotide primer combinations, methods or uses
described herein, the first polynucleotide (1) comprises a modified
nucleotide. In some aspects, the modified nucleotide is in domain (a),
and in further aspects the first polynucleotide (1) comprises a plurality
of modified nucleotides in domain (a). In another aspect, the modified
nucleotide is in domain (c) of the first polynucleotide (1).

[0017] The disclosure also provides polynucleotide primer combinations,
methods or uses in which the second polynucleotide (2) comprises a
modified nucleotide, and in some aspects the modified nucleotide is a
nucleotide at a 5' terminus or a 3' terminus of second polynucleotide
(2). Thus, in some embodiments, the modified nucleotide is the nucleotide
at the 3' terminus of second polynucleotide (2). In further aspects, the
modified nucleotide is in domain (f) and in some embodiments the second
polynucleotide (2) comprises a plurality of modified nucleotides in
domain (f). With respect to the modified nucleotide, the disclosure
provides embodiments wherein the modified nucleotide is the nucleotide
that is 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides from the 3' terminus of
the second polynucleotide (2).

[0018] It is generally known in the art how to synthesize polynucleotides
of various lengths. Accordingly, in various aspects the disclosure
provides polynucleotide primer combinations wherein domain (d) is from
about 5 bases in length to about 200 bases in length, about 5 bases in
length to about 150 bases in length, about 5 bases in length to about 100
bases in length, about 5 bases in length to about 50 bases in length,
about 5 bases in length to about 45 bases in length, about 5 bases in
length to about 40 bases in length, about 5 bases in length to about 35
bases in length, about 5 bases in length to about 30 bases in length,
about 5 bases in length to about 25 bases in length, about 5 bases in
length to about 20 bases in length, about 5 bases in length to about 15
bases in length, about 10 to about 50 bases in length, about 10 bases in
length to about 45 bases in length, about 10 bases in length to about 40
bases in length, about 10 bases in length to about 35 bases in length,
about 10 bases in length to about 30 bases in length, about 10 bases in
length to about 25 bases in length, about 10 bases in length to about 20
bases in length, or about 10 bases in length to about 15 bases in length.

[0019] In further aspects, polynucleotide primer combinations are provided
wherein domain (a) is from about 10 bases in length to about 5000 bases
in length, about 10 bases in length to about 4000 bases in length, about
10 bases in length to about 3000 bases in length, about 10 bases in
length to about 2000 bases in length, about 10 bases in length to about
1000 bases in length, about 10 bases in length to about 500 bases in
length, about 10 bases in length to about 250 bases in length, about 10
bases in length to about 200 bases in length, about 10 bases in length to
about 150 bases in length, about 10 bases in length to about 100 bases in
length, about 10 bases in length to about 95 bases in length, about 10
bases in length to about 90 bases in length, about 10 bases in length to
about 85 bases in length, about 10 bases in length to about 80 bases in
length, about 10 bases in length to about 75 bases in length, about 10
bases in length to about 70 bases in length, about 10 bases in length to
about 65 bases in length, about 10 bases in length to about 60 bases in
length, about 10 bases in length to about 55 bases in length, about 10
bases in length to about 50 bases in length, about 10 bases in length to
about 45 bases in length, about 10 bases in length to about 40 bases in
length, about 10 bases in length to about 35 bases in length, about 10
bases in length to about 30 bases in length, or about 10 bases in length
to about 100 bases in length.

[0020] In still further aspects, polynucleotide primer combinations are
provided wherein domain (c) is from about 5 bases in length to about 200
bases in length, about 5 bases in length to about 150 bases in length,
about 5 bases in length to about 100 bases in length, about 5 bases in
length to about 50 bases in length, about 5 bases in length to about 45
bases in length, about 5 bases in length to about 40 bases in length,
about 5 bases in length to about 35 bases in length, about 5 bases in
length to about 30 bases in length, about 5 bases in length to about 25
bases in length, about 5 bases in length to about 20 bases in length,
about 5 bases in length to about 15 bases in length, about 10 to about 50
bases in length, about 10 bases in length to about 45 bases in length,
about 10 bases in length to about 40 bases in length, about 10 bases in
length to about 35 bases in length, about 10 bases in length to about 30
bases in length, about 10 bases in length to about 25 bases in length,
about 10 bases in length to about 20 bases in length, or about 10 bases
in length to about 15 bases in length.

[0021] Polynucleotide primer combinations of the disclosure, in various
embodiments, are provided in which the first polynucleotide (1) and/or
the second polynucleotide (2) is DNA, modified DNA, RNA, modified RNA,
peptide nucleic acid (PNA), or combinations thereof.

[0022] In some aspects, polynucleotide primer combinations of the
disclosure further comprise an extension blocking group attached to the
first polynucleotide (1) at its 3' end which blocks extension from a DNA
polymerase. In various embodiments, the extension blocking group is
selected from the group consisting of a 3' phosphate group, a 3' amino
group, a dideoxy nucleotide, and an inverted deoxythymidine (dT).

[0024] Also provided are polynucleotide primer combinations wherein domain
(f) is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19 or 20 bases in length, and in one aspect domain (f) is 8 bases in
length. In some aspects, the sequence of domain (f) is 100% complementary
to the sequence of the second target polynucleotide region (F), while in
further aspects the sequence of domain (f) comprises a mismatch with the
sequence of the non-target polynucleotide region (F*). In related
aspects, polynucleotide primer combinations are provided wherein the
mismatch in the sequence of domain (f) with respect to the sequence of
the non-target polynucleotide region (F*) is not a 3' base mismatch,
while in further aspects polynucleotide primer combinations are provided
wherein the mismatch in the sequence of domain (f) with respect to the
sequence of the non-target polynucleotide region (F*) is a 3' base
mismatch. In an aspect, the mismatch is a mutation in the target
polynucleotide, and in further embodiments the mutation in the target
polynucleotide is selected from the group consisting of an insertion, a
deletion, a substitution and an inversion. In some embodiments, region
(F) and region (F*) comprise an identical sequence. Thus, in some
aspects, the sequence of domain (f) is 100% complementary to the sequence
of region (F*).

[0025] The disclosure further provides a polynucleotide primer combination
further comprising a blocker polynucleotide that comprises a 5' terminus
and a 3' terminus.

[0026] In aspects involving a blocker polynucleotide, it is contemplated
that the blocker polynucleotide comprises a nucleotide sequence that is
sufficiently complementary to region (F) such that the blocker
polynucleotide will hybridize to all or part of region (F) under
appropriate conditions. In some embodiments, a polynucleotide primer
combination is provided wherein a nucleotide at the 3' end of the second
polynucleotide (2) and a nucleotide at the 5' terminus of the blocker
polynucleotide overlap. In further embodiments, a polynucleotide primer
combination is provided wherein the blocker polynucleotide has a sequence
that overlaps (f) over the entire length of (f). In every aspect of the
disclosure, the blocker polynucleotide is perfectly complementary to a
region of a non-target polynucleotide and not perfectly complementary to
a region of a target polynucleotide. Also in every aspect of the
disclosure, domain (f) is perfectly complementary to a region in a target
polynucleotide.

[0027] Polynucleotide primer combinations are also provided wherein the
nucleotide at the 3' end of the second polynucleotide (2) and the
nucleotide at the 5' terminus of the blocker polynucleotide are
different. In one aspect, an internal nucleotide of the second
polynucleotide (2) and an internal nucleotide of the blocker
polynucleotide overlap. Thus, in various embodiments, the nucleotide of
the second polynucleotide (2) that overlaps with the nucleotide of the
blocker polynucleotide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18 or 19 nucleotides upstream of the 3' end of the second
polynucleotide (2). In further aspects, more than one nucleotide of
domain (f) is different from more than one nucleotide of the blocker
polynucleotide.

[0028] In any of the polynucleotide primer combinations of the disclosure,
it is contemplated that the second polynucleotide (2), the first
polynucleotide (1), and/or the blocker polynucleotide comprises a
modified nucleotide.

[0029] The disclosure further provides polynucleotide primer combinations
wherein the blocker polynucleotide is sufficiently complementary to
hybridize under appropriate conditions to a third target polynucleotide
region (X), wherein region (X) is between region (A) and region (F) in
the target polynucleotide. In some aspects, region (X) comprises a
sequence that is at least about 1 nucleotide to about 100 kilobases in
length. In further embodiments, region (X) comprises a sequence that is
at least about 2, at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10, at least 11, at least 12,
at least 13, at least 14, at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, at least 25, at least 26, at least 27, at least 28, at least
29, at least 30, at least 31, at least 32, at least 33, at least 34, at
least 35, at least 36, at least 37, at least 38, at least 39, at least
40, at least 41, at least 42, at least 43, at least 44, at least 45, at
least 46, at least 47, at least 48, at least 49, at least 50, at least
51, at least 52, at least 53, at least 54, at least 55, at least 56, at
least 57, at least 58, at least 59, at least 60, at least 61, at least
62, at least 63, at least 64, at least 65, at least 66, at least 67, at
least 68, at least 69, at least 70, at least 71, at least 72, at least
73, at least 74, at least 75, at least 76, at least 77, at least 78, at
least 79, at least 80, at least 81, at least 82, at least 83, at least
84, at least 85, at least 86, at least 87, at least 88, at least 89, at
least 90, at least 91, at least 92, at least 93, at least 94, at least
95, at least 96, at least 97, at least 98, at least 99, at least 100, at
least 110, at least 120, at least 130, at least 140, at least 150, at
least 160, at least 170, at least 180, at least 190, at least 200, at
least 210, at least 220, at least 230, at least 240, at least 250, at
least 260, at least 270, at least 280, at least 290, at least 300, at
least 310, at least 320, at least 330, at least 340, at least 350, at
least 360, at least 370, at least 380, at least 390, at least 400, at
least 410, at least 420, at least 430, at least 440, at least 450, at
least 460, at least 470, at least 480, at least 490, at least 500, at
least 510, at least 520, at least 530, at least 540, at least 550, at
least 560, at least 570, at least 580, at least 590, at least 600, at
least 610, at least 620, at least 630, at least 640, at least 650, at
least 660, at least 670, at least 680, at least 690, at least 700, at
least 710, at least 720, at least 730, at least 740, at least 750, at
least 760, at least 770, at least 780, at least 790, at least 800, at
least 810, at least 820, at least 830, at least 840, at least 850, at
least 860, at least 870, at least 880, at least 890, at least 900, at
least 910, at least 920, at least 930, at least 940, at least 950, at
least 960, at least 970, at least 980, at least 990 nucleotides, or at
least 1, at least 10, at least 20, at least 30, at least 40, at least 50,
at least 60, at least 70, at least 80, at least 90 or at least 100 or
more kilobases in length.

[0030] A polynucleotide primer combination of the disclosure, in various
aspects, further comprises a first rigid frame blocker polynucleotide
(RF1) and a second rigid frame blocker polynucleotide (RF2). In these
aspects, polynucleotide (RF1) comprises a domain (q) that is sufficiently
complementary to a region (Xq) in a non-target polynucleotide to allow
hybridization between domain (q) and region (Xq) under appropriate
conditions, a domain (t) that is sufficiently complementary to a region
(Xt) in the non-target polynucleotide to allow hybridization between
domain (t) and region (Xt), and a domain (r) that is sufficiently
complementary to a domain (v) in polynucleotide (RF2) to allow
hybridization between domain (r) and domain (v) under appropriate
conditions; and wherein polynucleotide (RF2) comprises a domain (u) that
is sufficiently complementary to a region (Xu) in a non-target
polynucleotide to allow hybridization between domain (u) and region (Xu)
under appropriate conditions, a domain (w) that is sufficiently
complementary to a region (Xw) in the non-target polynucleotide to allow
hybridization between domain (w) and region (Xw), and domain (v) that is
sufficiently complementary to domain (r) in polynucleotide (RF1) to allow
hybridization between domain (r) and domain (v) under appropriate
conditions, and wherein when domain (q) is specifically hybridized to
region (Xq), and domain (t) is specifically hybridized to region (Xt),
and domain (u) is specifically hybridized to region (Xu), and domain (w)
is specifically hybridized to region (Xw), and domain (r) is specifically
hybridized to domain (v), and domain (a) is specifically hybridized to
region (A), domain (f) will not hybridize to region (F), and wherein
region (Xq), region (Xt), region (Xu), and region (Xw) are not in the
target polynucleotide.

[0031] In various aspects of the disclosure, a polynucleotide primer
combination is provided wherein the blocker polynucleotide is
sufficiently complementary to hybridize under appropriate conditions to a
fourth target polynucleotide region (Y), wherein region (Y) is 5' of
region (F) in the target polynucleotide. In further embodiments, the
blocker polynucleotide is from at least about 1 to about 100 nucleotides
or more in length. In various aspects, the blocker polynucleotide is at
least about 2, at least 3, at least 4, at least 5, at least 6, at least
7, at least 8, at least 9, at least 10, at least 11, at least 12, at
least 13, at least 14, at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, at least 25, at least 26, at least 27, at least 28, at least
29, at least 30, at least 31, at least 32, at least 33, at least 34, at
least 35, at least 36, at least 37, at least 38, at least 39, at least
40, at least 41, at least 42, at least 43, at least 44, at least 45, at
least 46, at least 47, at least 48, at least 49, at least 50, at least
51, at least 52, at least 53, at least 54, at least 55, at least 56, at
least 57, at least 58, at least 59, at least 60, at least 61, at least
62, at least 63, at least 64, at least 65, at least 66, at least 67, at
least 68, at least 69, at least 70, at least 71, at least 72, at least
73, at least 74, at least 75, at least 76, at least 77, at least 78, at
least 79, at least 80, at least 81, at least 82, at least 83, at least
84, at least 85, at least 86, at least 87, at least 88, at least 89, at
least 90, at least 91, at least 92, at least 93, at least 94, at least
95, at least 96, at least 97, at least 98, at least 99, at least 100 or
more nucleotides in length. In one aspect, the blocker polynucleotide is
about 20 bases in length.

[0032] In some aspects of the disclosure, polynucleotide primer
combinations are provided that further comprise a probe polynucleotide,
the probe polynucleotide comprising a nucleotide sequence that is
complementary to the fourth target polynucleotide region (Y). In some
embodiments, the probe polynucleotide comprises a label and a quencher.
In any of the embodiments comprising a probe polynucleotide, it is
contemplated that the first polynucleotide, the second polynucleotide
and/or the probe polynucleotide comprises a modified nucleotide.

[0033] In certain polynucleotide primer combinations of the disclosure,
region (A) is 3' to region (F) in the target polynucleotide, while in
other polynucleotide primer combinations, region (A) is 5' to region (F)
in the target polynucleotide.

[0034] In further embodiments, a polynucleotide primer combination is
provided wherein at least one nucleotide in domain (a) overlaps at least
one nucleotide in domain (f). In some aspects, a polynucleotide primer
combination of the disclosure is provided wherein domain (a) comprises a
label. In an aspect, the label is quenchable. Accordingly, in certain
aspects a polynucleotide primer combination is provided wherein domain
(a) comprises a quencher. In specific embodiments, the quencher is
selected from the group consisting of Black Hole Quencher 1, Black Hole
Quencher-2, Iowa Black FQ, Iowa Black RQ, a G-Base, and Dabcyl.

[0035] A polynucleotide primer combination is also provided that further
comprises a reverse primer, wherein the reverse primer comprises a
polynucleotide sequence complementary to a sequence extended from domain
(f).

[0036] The disclosure further provides a method of detecting a target
polynucleotide in a sample with a primer combination, the primer
combination comprising a first polynucleotide (1) and a second
polynucleotide (2), the first polynucleotide (1) comprising a first
domain (a) having a sequence that is sufficiently complementary to a
first target polynucleotide region (A), a second domain (c) comprising a
unique polynucleotide sequence, and a third domain (b) comprising a
sequence that is not sufficiently complementary to hybridize to a domain
in the first polynucleotide (1), a domain in the second polynucleotide
(2), or a domain in the target polynucleotide, or the third domain
comprises a chemical polymer, the second polynucleotide (2) comprising a
first domain (f) that is fully complementary to a second target
polynucleotide region (F), a second domain (d) comprising a
polynucleotide sequence sufficiently complementary to domain (c) such
that domain (c) and domain (d) will hybridize under appropriate
conditions, and a third domain (e) comprising a sequence that is not
sufficiently complementary to hybridize to a domain in the first
polynucleotide (1), a domain in the second polynucleotide (2), or a
domain in the target polynucleotide, domain (f) having a sequence that is
not fully complementary to a non-target polynucleotide in the sample and
the method comprising the steps of: contacting the sample with the primer
combination and a polymerase under conditions that allow extension of a
sequence from domain (f) which is complementary to the target
polynucleotide when the second target polynucleotide region (F) is
present in the sample and detecting the sequence extended from domain (f)
indicating the second target polynucleotide region (F) is present in the
sample.

[0037] In some aspects, the method provides a change in sequence detection
from a sample with a non-target polynucleotide region compared to
sequence detection from a sample with a second target polynucleotide
region (F).

[0038] Also provided is a method of detecting a target polynucleotide in a
sample with any of the polynucleotide primer combinations of the
disclosure wherein the second polynucleotide (2) comprises a first domain
that is fully complementary to region (F) and wherein domain (f) is not
fully complementary to a non-target polynucleotide region in the sample,
the method comprising the steps of: contacting the sample with the primer
combination and a polymerase under conditions that allow extension of a
sequence from domain (f) which is complementary to the target
polynucleotide when the second target polynucleotide region (F) is
present in the sample and detecting the sequence extended from domain
(f). In various embodiments, the method provides a change in sequence
detection from a sample with a non-target polynucleotide region compared
to sequence detection from a sample with a second target polynucleotide
region (F).

[0039] In each of these methods, an embodiment is provided wherein the
detecting step is carried out using polymerase chain reaction. In these
embodiments, aspects are provided wherein the polymerase chain reaction
utilizes the second polynucleotide (2) of the primer combination and a
reverse primer, the reverse primer having a sequence complementary to the
sequence extended from domain (f). In further aspects, the polymerase
chain reaction utilizes a reverse primer complementary to the sequence
extended from domain (f) and a forward primer having a sequence
complementary to the strand of the target polynucleotide to which domain
(f) hybridizes.

[0040] In another aspect of these methods, detection is carried out in
real time.

[0041] The disclosure further provides a method of initiating polymerase
extension using a primer combination and a target polynucleotide as
template in a sample, the primer combination comprising a first
polynucleotide (1) and a second polynucleotide (2), the first
polynucleotide (1) comprising a first domain (a) having a sequence that
is sufficiently complementary to a first target polynucleotide region
(A), a second domain (c) comprising a unique polynucleotide sequence, and
a third domain (b) comprising a sequence that is not sufficiently
complementary to hybridize to a domain in the first polynucleotide (1), a
domain in the second polynucleotide (2), or a domain in the target
polynucleotide, or the third domain comprises a chemical polymer, the
second polynucleotide (2) comprising a first domain (f) that is fully
complementary to a second target polynucleotide region (F), a second
domain (d) comprising a polynucleotide sequence sufficiently
complementary to domain (c) such that domain (c) and domain (d) will
hybridize under appropriate conditions, and a third domain (e) comprising
a sequence that is not sufficiently complementary to hybridize to a
domain in the first polynucleotide (1), a domain in the second
polynucleotide (2), or a domain in the target polynucleotide, domain (f)
having a sequence that is not fully complementary to a non-target
polynucleotide in the sample, and wherein the sample comprises a mixture
of (i) a target polynucleotide that has a sequence (F) that is fully
complementary to the sequence in domain (f) and (ii) a non-target
polynucleotide that has a sequence (F*) that is not fully complementary
to (f), wherein the sequence of (F) is identical to the sequence of (F*)
except for at least a one nucleotide difference, the method comprising
the step of contacting the sample with the primer combination and a
polymerase under conditions that allow extension of a sequence from
domain (f) and complementary to the target polynucleotide strand when
domain (f) contacts region (F). In some aspects of methods of the
disclosure, the sequence in the region (F) in the target polynucleotide
differs from the sequence in the region (F*) in the non-target
polynucleotide at least at one base position. In a further aspect, a
method is provided further comprising the step of detecting the sequence
extended from domain (f), wherein detection indicates the presence of the
target polynucleotide in the sample.

[0042] In one aspect of any of the methods described herein, extension of
a sequence from domain (f) causes displacement of domain (a) from the
target polynucleotide. In another aspect, extension of a sequence from
domain (f) causes degradation of domain (a).

[0043] Also provided is a method of initiating polymerase extension using
any of the polynucleotide primer combinations described herein and a
target polynucleotide as template in a sample, wherein the second
polynucleotide (2) comprises a first domain (f) that is fully
complementary to a first target polynucleotide region (F) and wherein
domain (f) is not fully complementary to a non-target polynucleotide in
the sample, the method comprising the steps of: contacting the sample
with the primer combination and a polymerase under conditions that allow
extension of a sequence from domain (f) which is complementary to the
target polynucleotide when the target polynucleotide is present in the
sample. In some aspects, methods described herein further comprise the
step of detecting the sequence extended from domain (f), indicating the
presence of the target polynucleotide in the sample.

[0044] The disclosure further provides a method of amplifying a target
polynucleotide in a sample using a polynucleotide primer combination, the
primer combination comprising a first polynucleotide (1) and a second
polynucleotide (2), the first polynucleotide (1) comprising a first
domain (a) having a sequence that is sufficiently complementary to a
first target polynucleotide region (A), a second domain (c) comprising a
unique polynucleotide sequence, and a third domain (b) comprising a
sequence that is not sufficiently complementary to hybridize to a domain
in the first polynucleotide (1), a domain in the second polynucleotide
(2), or a domain in the target polynucleotide, or the third domain
comprises a chemical polymer, the second polynucleotide (2) comprising a
first domain (f) that is fully complementary to a second target
polynucleotide region (F), a second domain (d) comprising a
polynucleotide sequence sufficiently complementary to domain (c) such
that domain (c) and domain (d) will hybridize under appropriate
conditions, and a third domain (e) comprising a sequence that is not
sufficiently complementary to hybridize to a domain in the first
polynucleotide (1), a domain in the second polynucleotide (2), or a
domain in the target polynucleotide, domain (f) having a sequence that is
not fully complementary to a non-target polynucleotide in the sample and
wherein the sample comprises a mixture of (i) a target polynucleotide
that has a sequence in region (F) that is fully complementary to the
sequence in domain (f) and (ii) one or more non-target polynucleotides
that are not fully complementary to domain (f); the method comprising the
steps of: (a) contacting the sample with the primer combination and a
polymerase under conditions that allow extension of a sequence from
domain (f) which is complementary to the target polynucleotide when the
target polynucleotide is present in the sample, (b) denaturing the
sequence extended from domain (f) from the target polynucleotide, and (c)
repeating step (a) in the presence of a reverse primer having a sequence
complementary to a region in the sequence extended from domain (f) in
step (b) to amplify the target polynucleotide, wherein extension and
amplification of the target polynucleotide occurs when region (F) is
fully complementary to the sequence in the domain (f) but is less
efficient or does not occur when region (F) in the target polynucleotide
is not fully complementary to the sequence in domain (f).

[0045] Also provided is a method of amplifying a target polynucleotide in
a sample using any of the polynucleotide primer combinations of the
disclosure, wherein the second polynucleotide (2) comprises a first
domain (f) that is fully complementary to a first target polynucleotide
region (F) and wherein domain (f) is not fully complementary to a
non-target polynucleotide in the sample, the method comprising the steps
of: (a) contacting the sample with the primer combination and a
polymerase under conditions that allow extension of a sequence from
domain (f) which is complementary to the target polynucleotide when the
target polynucleotide is present in the sample, (b) denaturing the
sequence extended from domain (f) from the target polynucleotide, and (c)
repeating step (a) in the presence of a reverse primer having a sequence
complementary to a region in the sequence extended from (f) in step (b)
to amplify the target polynucleotide, wherein extension and amplification
of the target polynucleotide occurs when region (F) is fully
complementary to the sequence in domain (f) but is less efficient or does
not occur when the first region in the target polynucleotide is not fully
complementary to the sequence in domain (f). In some aspects, the reverse
primer has a sequence that is fully complementary to a region in the
sequence extended from domain (f), while in further aspects the reverse
primer is a primer combination comprising a first polynucleotide (3) and
a second polynucleotide (4), the first polynucleotide (3) comprising a
first domain (a2) having a sequence that is sufficiently complementary to
a first region (A2) in the sequence extended from domain (f) in step (a),
a second domain (c2) comprising a unique polynucleotide sequence, and a
third domain (b2) comprising a sequence that is not sufficiently
complementary to hybridize to a domain in (3), a domain in (4), a domain
in the first polynucleotide (1), a domain in the second polynucleotide
(2), a domain in the target polynucleotide or a domain in the sequence
extended from domain (f), or the third domain comprises a chemical
polymer, the second polynucleotide (4) comprising a first domain (f2)
that is fully complementary to a second region (F2) in the sequence
extended from domain (f) in step (a), a second domain (d2) comprising a
polynucleotide sequence sufficiently complementary to domain (c2) such
that domain (c2) and domain (d2) will hybridize under appropriate
conditions, and a third domain (e2) comprising a sequence that is not
sufficiently complementary to hybridize to a domain in (3), a domain in
(4), a domain in the first polynucleotide (1), a domain in the second
polynucleotide (2), a domain in the target polynucleotide or a domain in
the sequence extended from domain (f).

[0046] It will be understood that any of the polynucleotide primer
combinations disclosed herein may be used in any of the methods likewise
disclosed herein. Accordingly, in each method of the disclosure, an
aspect is provided wherein the reverse primer is a primer combination as
disclosed herein.

[0047] In each of the methods of the disclosure, the method further
comprises the step of detecting a product amplified in the method. In
some aspects, detection is carried out using polymerase chain reaction,
and in further aspects detection is carried out in real time. In some
embodiments, detection is carried out through the use of a detectable
marker.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1 is a schematic representing a primer combination of the
disclosure.

[0049] FIG. 2 is a schematic representing a primer combination in which
the relative positioning of domain (a) and domain (f) is inverted.

[0050] FIG. 3 is a schematic of mutant allele selection and extension by a
polynucleotide primer combination of the disclosure for use in detecting
a mutation in a target polynucleotide versus a non-target polynucleotide.

[0051] FIG. 4 depicts target polynucleotide discrimination with a
polynucleotide primer combination of the disclosure and a blocker
polynucleotide.

[0052] FIG. 5 depicts three forward polynucleotide primer combinations of
the disclosure for multiplexed PCR that have flexible domains (e2), (e4)
and (e6), each with the same universal sequence `e`.

[0053] FIG. 6 is a depiction of PCR with a polynucleotide primer
combination of the disclosure and a conventional (standard) reverse
primer.

[0054] FIG. 7 depicts the steps of PCR with a polynucleotide primer
combination of the disclosure and a conventional reverse primer.

[0055] FIG. 8 is a depiction of PCR with two polynucleotide primer
combinations of the disclosure.

[0056] FIG. 9 depicts a detailed protocol of PCR with two polynucleotide
primer combinations of the disclosure.

[0057] FIG. 10 depicts a polynucleotide primer combination of the
disclosure that further comprises a replication blocking domain (g).

[0058] FIG. 11 depicts PCR with two polynucleotide primer combinations of
the disclosure, each comprising a replication-blocking domain (g), and
two conventional primers.

[0059] FIG. 12 depicts a detailed protocol of PCR with two polynucleotide
primer combinations of the disclosure, each comprising a
replication-blocking domain (g), and two conventional primers.

[0060] FIG. 13 depicts a polynucleotide primer combination of the
disclosure in which region (A) is 5' to region (F) in the target
polynucleotide, for use in detecting a mutation in a target
polynucleotide versus a non-target polynucleotide.

[0061] FIG. 14 depicts a polynucleotide primer combination of the
disclosure in which region (A) is 5' to region (F) in the target
polynucleotide, for use in detecting a mutation in a target
polynucleotide versus a non-target polynucleotide wherein at least a
portion of domain (a) acts as a blocker polynucleotide.

[0062] FIG. 15 depicts a polynucleotide primer combination wherein domain
(a) further comprises a detectable marker and a quencher that quenches
the detectable marker, for use in detecting a mutation in a target
polynucleotide versus a non-target polynucleotide.

[0063] FIG. 16 depicts a polynucleotide primer combination wherein domain
(a) further comprises a detectable marker and a quencher that quenches
the detectable marker, and wherein domain (a) further comprises a blocker
polynucleotide domain (a5'), for use in detecting a mutation in a
target polynucleotide versus a non-target polynucleotide.

[0064] FIG. 17 depicts a polynucleotide primer combination that further
comprises a replication blocker domain within the second polynucleotide
(2).

[0065] FIG. 18 depicts a PCR method using a polynucleotide primer
combination of the disclosure in which region (A) is 5' to region (F) in
the target polynucleotide, without a replication blocker domain.

[0066] FIG. 19 depicts a polynucleotide primer combination of the
disclosure in which region (A) is 5' to region (F) in the target
polynucleotide and is used for PCR wherein the second polynucleotide (2)
and the fourth polynucleotide (4) comprise a replication blocker domain
(g).

[0067] FIG. 20 depicts the steps of PCR when using polynucleotide primer
combinations of the disclosure in which region (A) is 5' to region (F) in
the target polynucleotide, and wherein the second and fourth
polynucleotides (2) and (4) each comprise a replication blocker domain
(g).

[0068] FIG. 21 depicts potential interactions between polynucleotide
primer combinations of the disclosure in which region (A) is 5' to region
(F) in the target polynucleotide, wherein the second polynucleotide (2)
and the fourth polynucleotide (4) further comprise a replication blocker
domain (h) at different temperatures, and illustrates the lack of
amplifiable primer-dimer formation by the depicted polynucleotide primer
combinations.

[0069] FIG. 22 depicts polynucleotide primer combinations of the
disclosure in which region (A) is 5' to region (F) in the target
polynucleotide, wherein the second polynucleotide (2) and fourth
polynucleotide (4) further comprise a replication blocker domain (h) and
their use in PCR.

[0070] FIG. 23 A-G depicts the steps of PCR when using polynucleotide
primer combinations of the disclosure in which region (A) is 5' to region
(F) in the target polynucleotide, wherein the second and fourth
polynucleotides (2) and (4) each comprise a replication blocker domain
(h).

[0071] FIG. 24 depicts a polynucleotide primer combination in which the
first polynucleotide (1) comprises a self-blocking domain (j).

[0072] FIG. 25 depicts a polynucleotide primer combination in which the
first polynucleotide (1) comprises a self-blocking domain (j) and a
domain (p).

[0073] FIG. 26 shows a polynucleotide primer combination for detecting a
small deletion.

[0074] FIG. 27 shows a polynucleotide primer combination for detecting a
small deletion, and further comprising a blocker polynucleotide.

[0075] FIG. 28 depicts a polynucleotide primer combination for detecting a
large deletion and further comprising a blocker polynucleotide that
prevents looping-out and priming of non-target polynucleotide.

[0076] FIG. 29 depicts a rigid frame blocker polynucleotide and its
interaction with a non-target polynucleotide.

[0077] FIG. 30 shows the inhibition of non-target polynucleotide extension
by a polynucleotide primer combination of the disclosure using a rigid
frame blocker polynucleotide, wherein the deletion is at least 50 bases
in length.

[0093] The disclosure is based on the discovery of a discontinuous
polynucleotide design that overcomes problems encountered during the
hybridization of polynucleotides, and in particular, amplification primer
hybridization to a target polynucleotide. These problems include but are
not limited to a low specificity to single-base changes in a target
polynucleotide.

[0094] Polynucleotide combinations described herein offer an advantage
over both standard PCR primers and long PCR primers when using
polynucleotide templates that are difficult to amplify efficiently. Such
templates include, for example, those that contain a degree of secondary
structure formed through internal self-hybridization giving rise to, for
example, loops, hairpins and the like, that preclude, cause to be less
efficient or inhibit hybridization to a complementary sequence. Template
secondary structure can prevent priming with a standard PCR primer which
is unable to destabilize the internal hybridization and thus is unable to
hybridize to the primer complement. Using polynucleotide combinations of
the disclosure, template secondary structure is dehybridized (or melted)
and hybridization with the complementary template regions occurs under
appropriate conditions.

[0095] A long PCR primer is able to resolve secondary structure in a
target polynucleotide, but is not able to simultaneously provide either
the specificity or sensitivity near the 3' (priming) end of the primer.
This is because for a long PCR primer a large portion is hybridized to
the target polynucleotide and a mismatch near the 3' end of the primer
relative to the target polynucleotide will not be sufficient to reduce
priming efficiency. As a result, a PCR product will still be synthesized
despite the mismatch(s).

[0096] The polynucleotide combinations of the disclosure offer other
advantages. For example, short PCR primers alone are useful for precise
sequence hybridization to the target polynucleotide, but in order to
achieve the high specificity of primer binding to a target polynucleotide
that is desired for PCR, the highest possible annealing temperature is
typically chosen. This annealing temperature is chosen based on the
melting temperature of a given primer, and for a short primer that
annealing temperature will be relatively low. A low annealing
temperature, however, has the disadvantage of allowing for non-specific
hybridization of the short primer to the target polynucleotide, resulting
in non-specific PCR product formation. Based on the relatively low
annealing temperature that must be used to allow a short PCR primer to
anneal to its target polynucleotide, short primers form duplexes with a
target polynucleotide that are typically unstable even when they are 100%
complementary to the target polynucleotide region. Moreover, these
duplexes are even more unstable when the primer is less than 100%
complementary (i.e., at least one mismatch between the primer and the
target polynucleotide region). The polynucleotide combination of the
disclosure helps to overcome the instability problem associated with
using a short PCR primer and permit highly specific binding to a desired
target. For example, combinations of the disclosure are able to
discriminate between target sequences that differ by as little as a
single base.

[0097] For example, the polynucleotide combination design described herein
allows for use of a short primer domain (f) through hybridization of the
first domain (a) of the fixer polynucleotide (i.e., "first polynucleotide
(1)") to the target polynucleotide and hybridization of the second domain
[c] of the fixer polynucleotide (i.e., "first polynucleotide (1)") to the
second domain [d] of the primer polynucleotide (i.e., "second
polynucleotide (2)"), thereby giving the effective result of an apparent
"longer" primer sequence. This longer and discontinuous hybridization in
effect stabilizes binding between the first domain (f) of the second
polynucleotide (2) even if this region is as small as eight bases,
thereby increasing the efficiency of PCR. In another embodiment, the
regions of the target polynucleotide that are complementary to the first
domain of the first polynucleotide (1) and second polynucleotide (2) need
not be directly adjacent. The present disclosure provides polynucleotide
primers that comprise domain (b) in the second polynucleotide (2) and
domain (e) in the first polynucleotide (1) that allow the first domain
(a) of the first polynucleotide (1) and the first domain (f) of the
second polynucleotide (2) to "search" for their complements in the target
polynucleotide. This allows the distance between region (A) and region
(F) in the target polynucleotide to vary substantially. The flexible
linker domains cannot hybridize to a sequence in the first polynucleotide
(1), the second polynucleotide (2) or the target polynucleotide under
conditions in which domain (c) specifically hybridizes to domain (d).

TERMS

[0098] As used herein, a "standard" or "conventional" PCR primer is one
that hybridizes over its entire length to a polynucleotide.

[0099] The term "domain" as used herein refers to a contiguous sequence on
a polynucleotide primer of the disclosure. The term "region" as used
herein refers to a contiguous or non-contiguous sequence on a target or
non-target polynucleotide. The term "target polynucleotide" as used
herein refers to a polynucleotide from which extension by a polymerase is
desired, or to which a polynucleotide primer combination of the
disclosure is intended to hybridize. Thus, a "non-target polynucleotide"
is a polynucleotide from which extension by a polymerase is not desired,
or is less desirable than that of a target polynucleotide, or to which a
polynucleotide primer combination of the disclosure is intended to
hybridize with less specificity than a target polynucleotide.

[0100] "Appropriate conditions" as used herein refers to those conditions
that are determined by one of ordinary skill in the art, and generally
refer to nucleic acid hybridization conditions. One of skill in the art
will understand that "appropriate conditions" with respect to
hybridization depend on factors including but not limited to length of a
polynucleotide, relative G+C content, salt concentration and
hybridization temperature. Additional hybridization conditions are
discussed herein below.

[0101] "Specifically hybridize" as used herein means that a polynucleotide
will hybridize substantially or only with a specific nucleotide sequence
or a group of specific nucleotide sequences under stringent hybridization
conditions when the sequence is present in a complex mixture of DNA or
RNA. Stringent hybridization conditions are described herein below. One
or more nucleic acids are said to be "sufficiently complementary" when,
given a certain set of hybridizing conditions, the one or more nucleic
acids hybridize to each other. Accordingly, one or more nucleic acids are
said to be "not sufficiently complementary" when, given a certain set of
hybridizing conditions, the one or more nucleic acids do not hybridize to
each other. "Fully complementary" or "perfectly complementary" as used
herein means that a polynucleotide is 100% complementary to another
polynucleotide.

[0102] A "mutation" as used herein refers to one or more nucleotides in a
polynucleotide that differ from one or more corresponding nucleotides in
a wild-type polynucleotide. Examples of a mutation include but are not
limited to an insertion, a deletion, a substitution and an inversion. In
various aspects of the disclosure, a mutation is in a target
polynucleotide, with a wild-type sequecne being in a non-target
polynucleotide.

[0103] As used herein, a "chemical polymer" sequence is all or in part a
non-nucleic acid sequence that can be incorporated into a polynucleotide,
but cannot hybridize to a nucleic acid. In one aspect, the chemical
polymer is hydrophilic. Chemical polymers contemplated by the disclosure
include but are not limited to polyethylene glycol (PEG), a peptide and a
polysaccharide.

[0104] An "abasic site" as used herein is a Apurinic/Apyrimidinic (AP)
site in a nucleic acid sequence or a chemical polymer that can be
recognized and cleaved by an endonuclease.

[0105] A polynucleotide is said to "overlap" with another polynucleotide
when one or more bases of each polynucleotide can hybridize to the same
one or more bases of a target or non-target polynucleotide. By way of
example, where a first polynucleotide is complementary to a region in a
target nucleic acid and a second polynucleotide is complementary to all
or part of the same region in the target polynucleotide, the first
polynucleotide and the second polynucleotide are said to overlap.

[0106] A "unique polynucleotide sequence" as used herein refers to a
sequence in a polynucleotide primer that is not complementary to a
sequence in either a polynucleotide primer, a target polynucleotide or a
non-target polynucleotide.

[0107] Percent complementarity or "% complementary" as used herein refers
to a relative number of bases in a polynucleotide that are complementary
to a number of bases in another polynucleotide. Thus, in one non-limiting
example, if 18 out of 20 nucleotides in a polynucleotide primer of the
disclosure are perfectly complementary to a target polynucleotide, the
polynucleotide primer is said to be 90% complementary to the target
polynucleotide.

[0108] It is noted here that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise.

I. Polynucleotide Primer Combinations

[0109] In one embodiment, the present disclosure provides a polynucleotide
primer combination comprising a first polynucleotide (1) and a second
polynucleotide (2), the first polynucleotide (1) comprising a first
domain (a) having a sequence that is sufficiently complementary to a
first target polynucleotide region (A), a second domain (c) comprising a
unique polynucleotide sequence, and a third domain (b) comprising a
polymer sequence that is not sufficiently complementary to hybridize to a
domain in the first polynucleotide (1), a domain in the second
polynucleotide (2), or a domain in the target polynucleotide, wherein
domains in the first polynucleotide are arranged 5'-a-b-c-3'; the second
polynucleotide (2) comprising a first domain (f) having a sequence that
is sufficiently complementary to a sequence in a second target
polynucleotide region (F), a second domain (d) comprising a
polynucleotide sequence sufficiently complementary to (c) such that (c)
and (d) will hybridize under appropriate conditions, and a third domain
(e) comprising a polynucleotide sequence that is not sufficiently
complementary to hybridize to a domain in the first polynucleotide (1), a
domain in the second polynucleotide (2), or a domain in the target
polynucleotide, wherein domains in the second polymer are arranged
5'-d-e-f-3', wherein under conditions in which region (A) specifically
hybridizes to domain (a) and region (F) specifically hybridizes to domain
(f), domain (c) hybridizes to domain (d) and neither domain (b) nor
domain (e) hybridizes to a domain in the first polynucleotide (1), a
domain in second polynucleotide (2) or a domain in the target
polynucleotide (see FIG. 1).

[0110] In some embodiments, the polymer sequence of domain (b) is selected
from the group consisting of a polynucleotide sequence, a polynucleotide
sequence comprising at least one modified nucleotide, a
non-polynucleotide chemical polymer sequence and combinations thereof. In
further embodiments, the polynucleotide sequence of domain (e) is
selected from the group consisting of a polynucleotide sequence of
naturally-occurring nucleotides and a polynucleotide sequence comprising
at least one modified nucleotide, wherein the polynucleotide sequence of
domain (e) is a template for polymerase extension.

[0111] In another embodiment, a polynucleotide primer combination is
provided further comprising a domain (g) positioned between domain (d)
and domain (e) in the second polynucleotide, wherein domain (g) comprises
a replication blocker. In a further embodiment, a polynucleotide primer
combination is provided wherein the second polynucleotide further
comprises a fourth domain (h) positioned between domain (f) and domain
(e), wherein domain (h) comprises a replication blocker. In various
aspects, the replication blocker is selected from the group consisting of
a modified base, an abasic site and a polymer.

[0112] In some aspects, a polynucleotide primer combination is provided
wherein the second polynucleotide (2) further comprises a domain (s)
positioned 5' of domain (d), wherein domain (s) comprises a sequence that
is not sufficiently complementary to the first polynucleotide (1) or the
second polynucleotide (2) to hybridize to the first polynucleotide (1) or
the second polynucleotide (2) under conditions in which domain (a)
specifically hybridizes to region (A), and optionally wherein domain (s)
is sufficiently complementary to a polynucleotide extended from domain
(f) to hybridize to the polynucleotide extended from domain (f) under
conditions in which domain (a) specifically hybridizes to region (A). In
various embodiments, domain (s) further comprises a detectable marker,
and in still further embodiments domain (s) further comprises a quencher
that quenches the detectable marker.

[0113] In another embodiment, a polynucleotide primer combination is
provided wherein domain (a) of the first polynucleotide (1) further
comprises a domain (j), wherein domain (j) is contiguous with domain (a)
and is positioned 3' of domain (a), and wherein domain (j) is
sufficiently complementary to a region (F*) in a non-target
polynucleotide to hybridize under conditions in which domain (a)
specifically hybridizes to region (A), wherein region (F*) in the
non-target polynucleotide differs from region (F) in the target
polynucleotide at at least one nucleotide. In a related embodiment, a
polynucleotide primer combination is provided wherein domain (a) and
domain (j) are not contiguous and are separated by a domain (p), wherein
domain (p) is not sufficiently complementary to a domain in the first
polynucleotide (1), a domain in the second polynucleotide (2), or the
target polynucleotide to hybridize to the first polynucleotide (1), the
second polynucleotide (2) or the target polynucleotide under conditions
wherein domain (a) specifically hybridizes to region (A).

[0114] In some aspects, a polynucleotide primer combination is provided
wherein domain (c) is at least 70% complementary to domain (d). In
another embodiment, a polynucleotide primer combination is provided
wherein domain (d) is at least 70% complementary to domain (c). With
respect to domain (c) and domain (d), it is contemplated that in some
aspects domain (d) and domain (c) are sufficiently complementary to
hybridize to each other in the absence of the template polynucleotide.

[0115] In another embodiment, a polynucleotide primer combination is
provided further comprising a blocking group attached to the first
polynucleotide at its 3' end which blocks extension from a DNA
polymerase. In various aspects, the blocking group is selected from the
group consisting of a 3' phosphate group, a 3' amino group, a dideoxy
nucleotide, and an inverted deoxythymidine (dT).

[0117] In some aspects, a polynucleotide primer combination is provided
wherein domain (f) is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19 or 20 bases in length. In one aspect, domain
(f) is 8 bases in length. It is also contemplated that in some
embodiments the sequence of domain (f) is 100% complementary to the
sequence of the second target polynucleotide region (F). However, in
certain aspects of the disclosure the sequence of domain (f) comprises a
mismatch with the sequence of the non-target polynucleotide region (F*).
In these aspects, the mismatch in the sequence of domain (f) with respect
to the sequence of the second target polynucleotide region (F) is not a
3' base mismatch, or the mismatch in the sequence of domain (f) with
respect to the sequence of the second target polynucleotide region (F) is
a 3' base mismatch. In some aspects, the mismatch is a mutation.

[0118] Accordingly, polynucleotide primer combinations of the disclosure
are useful in the highly specific detection of mutant sequences. In
various aspects, the mutation is selected from the group consisting of an
insertion, a deletion, a substitution and an inversion.

A. Primer Combinations Comprising a Blocker Polynucleotide

[0119] The disclosure contemplates embodiments wherein a blocker
polynucleotide is included with a polynucleotide combination. A blocker
polynucleotide comprises a nucleotide sequence that is sufficiently
complementary to all or part of region (F) such that the blocker
polynucleotide will hybridize to all or part of region (F) under
appropriate conditions. In some embodiments, the blocker polynucleotide
overlaps with the first domain (f) of the second polynucleotide (2). In
other words, the nucleotide(s) at the 3' end of the second polynucleotide
(2) and the nucleotide(s) at the 5' end of the blocker polynucleotide
would be complementary to the same nucleotide(s) of the target
polynucleotide. In some aspects, the blocker polynucleotide has a
sequence that overlaps (f) over the entire length of (f). In various
embodiments, the overlap of the second polynucleotide (2) and the blocker
polynucleotide is 1 nucleotide, 2 nucleotides, 3 nucleotides, 4
nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides,
9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13
nucleotides, 14 nucleotides, or 15 nucleotides. In related embodiments,
the nucleotide(s) at the 3' end of the second polynucleotide (2) and the
nucleotide(s) at the 5' end of the blocker polynucleotide are different.
In these embodiments, the nucleotide(s) at the 3' end of the second
polynucleotide (2) would hybridize to the target polynucleotide when they
are complementary to the target polynucleotides at the appropriate
position, thus allowing for extension of the second polynucleotide (2)
under the appropriate conditions. In related embodiments, the
nucleotide(s) at the 5' end of the blocker polynucleotide would not
hybridize to the target polynucleotide when the blocker polynucleotide is
perfectly complementary to the non-target polynucleotide at the
appropriate position, thus blocking extension of the second
polynucleotide (2) from the non-target polynucleotide. In various
embodiments, the nucleotide at the 3' end of the blocker polynucleotide
is modified to prevent extension by a polymerase.

[0120] In some embodiments, the overlapping sequences of the blocker
polynucleotide and the first domain (f) of the second polynucleotide (2)
differ by at least 2 bases, at least 3 bases, at least 4 bases, at least
5 bases, at least 6 bases, at least 7 bases, at least 8 bases, at 9 two
bases, or by at least 10 bases. The differing bases can be at any
position in the overlapping portions.

[0121] In further embodiments, the blocker polynucleotide is sufficiently
complementary to hybridize under appropriate conditions to a third target
polynucleotide region (X), wherein region (X) is between region (A) and
region (F). In various aspects, region (X) comprises a sequence that is
at least about 1 nucleotide to about 100 kilobases in length.

[0122] In some embodiments, a polynucleotide primer combination of the
disclosure further comprises a first rigid frame blocker polynucleotide
(RF1) and a second rigid frame blocker polynucleotide (RF2).
Polynucleotide (RF1) comprises a domain (q) that is sufficiently
complementary to a region (Xq) in a non-target polynucleotide to allow
hybridization between domain (q) and region (Xq) under appropriate
conditions, a domain (t) that is sufficiently complementary to a region
(Xt) in the non-target polynucleotide to allow hybridization to allow
hybridization between domain (t) and region (Xt), and a domain (r) that
is sufficiently complementary to a domain (v) in polynucleotide (RF2) to
allow hybridization between domain (r) and domain (v) under appropriate
conditions; and wherein polynucleotide (RF2) comprises a domain (u) that
is sufficiently complementary to a region (Xu) in a non-target
polynucleotide to allow hybridization between domain (u) and region (Xu)
under appropriate conditions, a domain (w) that is sufficiently
complementary to a region (Xw) in the non-target polynucleotide to allow
hybridization between domain (w) and region (Xw), and domain (v) that is
sufficiently complementary to domain (r) in polynucleotide (RF 1) to
allow hybridization between domain (r) and domain (v) under appropriate
conditions, and wherein when domain (q) is specifically hybridized to
region (Xq), and domain (t) is specifically hybridized to region (Xt),
and domain (u) is specifically hybridized to region (Xu), and domain (w)
is specifically hybridized to region (Xw), and domain (r) is specifically
hybridized to domain (v), and domain (a) is specifically hybridized to
region (A), domain (f) will not hybridize to region (F), and wherein
region (Xq), region (Xt), region (Xu) and region (Xw) are not in the
target polynucleotide.

[0123] In another aspect, the blocker polynucleotide is sufficiently
complementary to hybridize under appropriate conditions to a fourth
target polynucleotide region (Y), wherein region (Y) is 5' of region (F).

[0124] In further aspects, the blocker polynucleotide is sufficiently
complementary to hybridize under appropriate conditions to the entire
length of region (F) and, in additional aspects, is able to hybridize
under appropriate conditions either partially or entirely to region (A).

[0125] In some aspects, domain (b) of polynucleotide 1 is absent (i.e.,
comprises 0 bases), while in further aspects, domain (e) is absent (i.e.,
comprises 0 bases). In these aspects, it will be understood that in
aspects wherein domain (b) is absent, domain (e) is present. Conversely,
in aspects wherein domain (e) is absent, domain (b) is present.
Accordingly, the disclosure contemplates that either domain (b) or domain
(e) is absent in various aspects, but in no aspect are both domain (b)
and domain (e) simultaneously absent.

[0126] The blocker polynucleotide is, in various aspects, from at least
about 1 to at least about 100 bases or more in length. In one aspect, the
blocker polynucleotide is 20 bases in length.

B. Primer Combinations Comprising a Probe Polynucleotide

[0127] The disclosure also contemplates embodiments further comprising a
probe polynucleotide. In some embodiments, a probe polynucleotide
comprises a nucleotide sequence that is complementary to the fourth
target polynucleotide region (Y). In other embodiments, a probe
polynucleotide has a sequence that is complementary to the extension
product of the second polynucleotide. As is apparent, this probe
polynucleotide would be complementary to the complementary strand of the
target polynucleotide. In embodiments wherein a blocker polynucleotide is
included in the primer combination with the probe polynucleotide, the
probe polynucleotide is complementary to a target polynucleotide region
located 5' of the target polynucleotide region complementary to the
blocker polynucleotide. In various embodiments, the probe polynucleotide
comprises a label at its 5' end. In related embodiments, the probe
polynucleotide further comprises a quencher at its 3' end. In still
further embodiments, the probe polynucleotide further comprises an
internal quencher, such as, and without limitation, the Zen quencher.

C. Primer Combinations--Positioning of Domain (a) and Domain (f)

[0128] The disclosure provides polynucleotide primer combinations in which
the relative positioning of domain (a) and domain (f) is inverted (see
FIG. 2). Thus, in one aspect, a polynucleotide primer combination is
provided wherein region (A) is 3' to region (F) in the target
polynucleotide. In another aspect, a polynucleotide primer combination is
provided wherein region (A) is 5' to region (F) in the target
polynucleotide.

[0129] In aspects wherein region (A) is 5' to region (F), it is
contemplated that in some aspects, at least one nucleotide in domain (a)
overlaps at least one nucleotide in domain (f) when each are hybridized
to their respective region in the target polynucleotide.

[0130] In some aspects of the disclosure, domain (a) comprises a label,
and in further aspects the label is quenchable. Thus, in some aspects
domain (a) comprises a quencher, wherein the quencher is selected from
the group consisting of Black Hole Quencher 1, Black Hole Quencher-2,
Iowa Black FQ, Iowa Black RQ, a G-Base, and Dabcyl.

D. Primer Combinations Comprising a Reverse Primer

[0131] The disclosure also contemplates embodiments wherein a reverse
primer polynucleotide is included with the above polynucleotide
combinations. The reverse primer is complementary to a region in the
polynucleotide created by extension of the second polynucleotide. As is
apparent, in some embodiments the reverse primer is also complementary to
the complementary strand of the target polynucleotide when the target
polynucleotide is one strand of a double-stranded polynucleotide. In some
embodiments, the reverse primer is a combination first
polynucleotide/second polynucleotide, as described above.

II. Polynucleotides

[0132] As used herein, the term "polynucleotide," either as a component of
a polynucleotide pair combination, including blocker polynucleotides and
probes, or as a target molecule, is used interchangeably with the term
oligonucleotide and the term "nucleic acid."

[0133] The term "nucleotide" or its plural as used herein includes
naturally-occurring and modified forms as discussed herein and otherwise
known in the art. In certain instances, the art uses the term
"nucleobase" which embraces naturally-occurring nucleotides as well as
modifications of nucleotides that can be polymerized.

[0134] Methods of making polynucleotides of a predetermined sequence are
well-known in the art. See, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual (2nd ed. 1989) and F. Eckstein (ed.) Oligonucleotides
and Analogues, 1st Ed. (Oxford University Press, New York, 1991).
Solid-phase synthesis methods are preferred for both oligoribonucleotides
and oligodeoxyribonucleotides (the well-known methods of synthesizing DNA
are also useful for synthesizing RNA). Oligoribonucleotides and
oligodeoxyribonucleotides can also be prepared enzymatically.

[0135] In various aspects, methods provided include use of polynucleotides
which are DNA oligonucleotides, RNA oligonucleotides, or combinations of
the two types. Modified forms of oligonucleotides are also contemplated
which include those having at least one modified internucleotide linkage.
Modified polynucleotide also include a chemical polymer as used herein.
Modified polynucleotides or oligonucleotides are described in detail
herein below.

III. Modified Polynucleotides

[0136] Specific examples of oligonucleotides include those containing
modified backbones or non-natural internucleoside linkages.
Oligonucleotides having modified backbones include those that retain a
phosphorus atom in the backbone and those that do not have a phosphorus
atom in the backbone. Modified oligonucleotides that do not have a
phosphorus atom in their internucleoside backbone are considered to be
within the meaning of "oligonucleotide." In specific embodiments, the
first polynucleotide comprises phosphorothioate linkages.

[0137] Modified oligonucleotide backbones containing a phosphorus atom
include, for example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl
and other alkyl phosphonates including 3'-alkylene phosphonates,
5'-alkylene phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates
and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein one or more
internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.
Also contemplated are oligonucleotides having inverted polarity
comprising a single 3' to 3' linkage at the 3'-most internucleotide
linkage, i.e. a single inverted nucleoside residue which may be abasic
(the nucleotide is missing or has a hydroxyl group in place thereof).
Salts, mixed salts and free acid forms are also contemplated.
Representative United States patents that teach the preparation of the
above phosphorus-containing linkages include, U.S. Pat. Nos. 3,687,808;
4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;
5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;
5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;
5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599;
5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, the disclosures
of which are incorporated by reference herein.

[0139] In still other embodiments, a modified oligonucleotide includes
mimetics wherein both one or more sugar and/or one or more
internucleotide linkage of the nucleotide units are replaced with
"non-naturally occurring" groups. In one aspect, this embodiment
contemplates a peptide nucleic acid (PNA). In PNA compounds, the
sugar-backbone of an oligonucleotide is replaced with an amide containing
backbone. See, for example U.S. Pat. Nos. 5,539,082; 5,714,331; and
5,719,262, and Nielsen et al., 1991, Science, 254: 1497-1500, the
disclosures of which are herein incorporated by reference.

[0140] In still other embodiments, oligonucleotides are provided with
phosphorothioate backbones and oligonucleosides with heteroatom
backbones, and including --CH2--NH--O--CH2--,
--CH2--N(CH3)--O--CH2--,
--CH2--O--N(CH3)--CH2--,
--CH2--N(CH3)--N(CH3)--CH2-- and
--O--N(CH3)--CH2--CH2-- described in U.S. Pat. Nos.
5,489,677, and 5,602,240. Also contemplated are oligonucleotides with
morpholino backbone structures described in U.S. Pat. No. 5,034,506.

[0142] Still other modified forms of oligonucleotides are described in
detail in U.S. patent application NO. 20040219565, the disclosure of
which is incorporated by reference herein in its entirety.

[0143] Modified oligonucleotides may also contain one or more substituted
sugar moieties. In certain aspects, oligonucleotides comprise one of the
following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or
N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,
alkenyl and alkynyl may be substituted or unsubstituted C1 to
C10 alkyl or C2 to C10 alkenyl and alkynyl. Other
embodiments include O[(CH2)nO]mCH3,
O(CH2)nOCH3, O(CH2)nNH2,
O(CH2)nCH3, O(CH2)nONH2, and
O(CH2)nON[(CH2)mCH3]2, where n and m are
from 1 to about 10. Other oligonucleotides comprise one of the following
at the 2' position: C1 to C10 lower alkyl, substituted lower
alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,
SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3,
SO2CH3, ONO2, NO2, N3, NH2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,
substituted silyl, an RNA cleaving group, a reporter group, an
intercalator, a group for improving the pharmacokinetic properties of an
oligonucleotide, or a group for improving the pharmacodynamic properties
of an oligonucleotide, and other substituents having similar properties.
In one aspect, a modification includes 2'-methoxyethoxy
(2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or
2'-MOE) (Martin et al., 1995, Helv. Chim. Acta, 78: 486-504) i.e., an
alkoxyalkoxy group. Other modifications include
2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2
group, also known as 2'-DMAOE, as described in examples herein below, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e.,
2'-O--CH2--O--CH2--N(CH3)2, also described in
examples herein below.

[0144] Still other modifications include 2'-methoxy (2'-O--CH3),
2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-allyl
(2'-CH2--CH═CH2), 2'-O-allyl
(2'-O--CH2--CH═CH2) and 2'-fluoro (2'-F). The
2'-modification may be in the arabino (up) position or ribo (down)
position. In one aspect, a 2'-arabino modification is 2'-F. Similar
modifications may also be made at other positions on the oligonucleotide,
for example, at the 3' position of the sugar on the 3' terminal
nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5'
terminal nucleotide. Oligonucleotides may also have sugar mimetics such
as cyclobutyl moieties in place of the pentofuranosyl sugar. See, for
example, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;
5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;
5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;
5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, the
disclosures of which are incorporated by reference in their entireties
herein.

[0145] In various aspects, a modification of the sugar includes Locked
Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3'
or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar
moiety. The linkage in certain aspects is a methylene
(--CH2--)n group bridging the 2' oxygen atom and the 4' carbon
atom wherein n is 1 or 2. LNAs and preparation thereof are described in
WO 98/39352 and WO 99/14226, the disclosures of which are incorporated by
reference in their entireties herein. In various embodiments, the first
polynucleotide comprises a locked nucleic acid. In some embodiments, the
first polynucleotide comprises a plurality of locked nucleic acids. In
specific embodiments, the first domain of the first polynucleotide
comprises a plurality of locked nucleic acids. In more specific
embodiments, the nucleotide at the 3' end of the first polynucleotide
comprises a locked nucleic acid. In various embodiments, the blocker
polynucleotide comprises a locked nucleic acid. In other embodiments, the
blocker polynucleotide comprises a plurality of locked nucleic acids. In
specific embodiments, the nucleotide at the 5' end of the blocker
polynucleotide comprises a locked nucleic acid.

[0146] Polynucleotides may also include base modifications or
substitutions. As used herein, "unmodified" or "natural" bases include
the purine bases adenine (A) and guanine (G), and the pyrimidine bases
thymine (T), cytosine (C) and uracil (U). Modified bases include other
synthetic and natural bases such as 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and
other alkyl derivatives of adenine and guanine, 2-thiouracil,
2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl
uracil and cytosine and other alkynyl derivatives of pyrimidine bases,
6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other
8-substituted adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,
8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and
3-deazaguanine and 3-deazaadenine. Further modified bases include
tricyclic pyrimidines such as phenoxazine
cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine
cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such
as a substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzox-azin-2(3H)-one), carbazole
cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine
(H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases may
also include those in which the purine or pyrimidine base is replaced
with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine,
2-aminopyridine and 2-pyridone. Further bases include those disclosed in
U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of
Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.
John Wiley & Sons, 1990, those disclosed by Englisch et al., 1991,
Angewandte Chemie, International Edition, 30: 613, and those disclosed by
Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages
289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of
these bases are useful for increasing the binding affinity and include
5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6
substituted purines, including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown to
increase nucleic acid duplex stability by 0.6-1.2° C. and are, in
certain aspects combined with 2'-O-methoxyethyl sugar modifications. See,
U.S. Pat. No. 3,687,808, U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;
5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;
5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;
5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,750,692 and
5,681,941, the disclosures of which are incorporated herein by reference.

[0147] A "modified base" or other similar term refers to a composition
which can pair with a natural base (e.g., adenine, guanine, cytosine,
uracil, and/or thymine) and/or can pair with a non-naturally occurring
base. In certain aspects, the modified base provides a Tm
differential of 15, 12, 10, 8, 6, 4, or 2° C. or less. Exemplary
modified bases are described in EP 1 072 679 and WO 97/12896.

[0148] By "nucleobase" is meant the naturally occurring nucleobases
adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) as
well as non-naturally occurring nucleobases such as xanthine,
diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine,
7-deazaguanine, N4,N4-ethanocytosin,
N',N'-ethano-2,6-diaminopurine, 5-methylcytosine (mC),
5-(C3-C6)-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil,
pseudoisocytosine, 2-hydroxy-5-methyl-4-tr-iazolopyridin, isocytosine,
isoguanine, inosine and the "non-naturally occurring" nucleobases
described in Benner et al., U.S. Pat. No. 5,432,272 and Susan M. Freier
and Karl-Heinz Altmann, 1997, Nucleic Acids Research, vol. 25: pp
4429-4443. The term "nucleobase" thus includes not only the known purine
and pyrimidine heterocycles, but also heterocyclic analogues and
tautomers thereof. Further naturally and non-naturally occurring
nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan,
et al.), in Chapter 15 by Sanghvi, in Antisense Research and Application,
Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, in Englisch et al.,
1991, Angewandte Chemie, International Edition, 30: 613-722 (see
especially pages 622 and 623, and in the Concise Encyclopedia of Polymer
Science and Engineering, J. I. Kroschwitz Ed., John Wiley & Sons, 1990,
pages 858-859, Cook, Anti-Cancer Drug Design 1991, 6, 585-607, each of
which are hereby incorporated by reference in their entirety). The term
"nucleosidic base" or "base unit" is further intended to include
compounds such as heterocyclic compounds that can serve like nucleobases
including certain "universal bases" that are not nucleosidic bases in the
most classical sense but serve as nucleosidic bases. Especially mentioned
as universal bases are 3-nitropyrrole, optionally substituted indoles
(e.g., 5-nitroindole), and optionally substituted hypoxanthine. Other
desirable universal bases include, pyrrole, diazole or triazole
derivatives, including those universal bases known in the art.

IV. Polynucleotide Structure--Length

[0149] In one embodiment, the first polynucleotide (1) comprises a first
domain (a) containing about 10 nucleotides, this first domain (a) of the
first polynucleotide (1) being complementary to a target polynucleotide
region that is different from the target region recognized by the first
domain (f) of the second polynucleotide (2). In various aspects, the
first polynucleotide (1) comprises a first domain (a) containing at least
11, at least 12, at least 13, at least 14, at least 15, at least 16, at
least 17, at least 18, 19, at least 20, at least 21, at least 22, at
least 23, at least 24, at least 25, at least 26, at least 27, at least
28, at least 29, at least 30, at least 31, at least 32, at least 33, at
least 34, at least 35, at least 36, at least 37, at least 38, at least
39, at least 40, at least 41, at least 42, at least 43, at least 44, at
least 45, at least 46, at least 47, at least 48, at least 49, at least
50, at least about 100, at least about 150, at least about 200, at least
about 250, at least about 300, at least about 350, at least about 400, at
least about 450, at least about 500, at least about 550, at least about
600, at least about 650, at least about 700, at least about 750, at least
about 800, at least about 850, at least about 900, at least about 950, at
least about 1000, at least about 1100, at least about 1200, at least
about 1300, at least about 1400, at least about 1500, at least about
1600, at least about 1700, at least about 1800, at least about 1900, at
least about 2000, at least about 2100, at least about 2200, at least
about 2300, at least about 2400, at least about 2500, at least about
2600, at least about 2700, at least about 2800, at least about 2900, at
least about 3000, at least about 3100, at least about 3200, at least
about 3300, at least about 3400, at least about 3500, at least about
3600, at least about 3700, at least about 3800, at least about 3900, at
least about 4000, at least about 4100, at least about 4200, at least
about 4300, at least about 4400, at least about 4500, at least about
4600, at least about 4700, at least about 4800, at least about 4900, at
least about 5000 or more nucleotides, the first domain (a) of this first
polynucleotide (1) being complementary, or sufficiently complementary, so
as to recognize and bind to a target polynucleotide region that is
different from the target region recognized by the first domain (f) of
the second polynucleotide (2). In a related aspect, the second domain (d)
of the second polynucleotide (2) comprises 6 or more nucleotides in a
unique DNA polynucleotide sequence that is sufficiently complementary to
the second domain (c) of the first polynucleotide (1) so as to allow
hybridization between these two complementary sequences under appropriate
conditions. In a related aspect, the second domain (c) of the first
polynucleotide (1) comprises 6 or more nucleotides in a unique
polynucleotide sequence that is sufficiently complementary to the second
domain (d) of the second polynucleotide (2) so as to allow hybridization
between these two complementary sequences under appropriate conditions.
In various aspects, the second domain (c) of the first polynucleotide (1)
comprises at least 11, at least 12, at least 13, at least 14, at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24 nucleotides, at least 25,
at least about 30, at least about 35, at least about 40, at least about
45, at least about 50, at least about 60, at least about 70, at least
about 80, at least about 90, at least about 100, at least about 120, at
least about 140, at least about 160, at least about 180, at least about
200, at least about 220, at least about 240, at least about 260, at least
about 280, at least about 300, at least about 320, at least about 340, at
least about 360, at least about 380, at least about 400, at least about
420, at least about 440, at least about 460, at least about 480, at least
about 500 or more nucleotides of a unique polynucleotide sequence that is
sufficiently complementary to the second domain (d) of the second
polynucleotide (2) so as to allow hybridization between the two
complementary sequences under appropriate conditions.

[0150] In another aspect, the first domain (f) of the second
polynucleotide (2) is 2 nucleotides that are complementary to a target
polynucleotide region (F). In various aspects, the first domain (f) of
the second polynucleotide (2) is at least 3 nucleotides, at least 4
nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7
nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10
nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least
13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at
least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides,
at least 19 nucleotides, at least 20 nucleotides, at least 21
nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least
24 nucleotides, at least 25 nucleotides, at least 26 nucleotides, at
least 27 nucleotides, at least 28 nucleotides, at least 29 nucleotides,
at least 30 nucleotides or more that is complementary to a target
polynucleotide region (F). In a related aspect, the second domain (d) of
the second polynucleotide (2) comprises 10 or more nucleotides in a
unique polynucleotide sequence that is sufficiently complementary to the
second domain (c) of the first polynucleotide (1) so as to allow
hybridization between these two complementary sequences under appropriate
conditions. In various aspects, the second domain (d) of the second
polynucleotide (2) comprises at least 11, at least 12, at least 13, at
least 14, at least 15, at least 16, at least 17, at least 18, at least
19, at least 20, at least 21, at least 22, at least 23, at least 24
nucleotides, at least 25, at least about 30, at least about 35, at least
about 40, at least about 45, at least about 50, at least about 60, at
least about 70, at least about 80, at least about 90, at least about 100,
at least about 120, at least about 140, at least about 160, at least
about 180, at least about 200, at least about 220, at least about 240, at
least about 260, at least about 280, at least about 300, at least about
320, at least about 340, at least about 360, at least about 380, at least
about 400, at least about 420, at least about 440, at least about 460, at
least about 480, at least about 500 or more nucleotides of a unique
polynucleotide sequence that is sufficiently complementary to the second
domain (c) of the first polynucleotide (1) so as to allow hybridization
between the two complementary sequences under appropriate conditions.

[0151] As described herein, the third domain (b) of the first
polynucleotide (1) comprises a polymer sequence is selected from the
group consisting of a polynucleotide sequence, a polynucleotide sequence
comprising at least one modified nucleotide, and a non-polynucleotide
chemical polymer sequence. In aspects wherein domain (b) is a
polynucleotide sequence, the polynucleotide sequence is, in various
aspects, at least 1 nucleotide in length. Similarly, domain (e) of the
second polynucleotide is, in some aspects, at least 1 nucleotide in
length and can comprise a polynucleotide sequence of naturally-occurring
nucleotides and a polynucleotide sequence comprising at least one
modified nucleotide. In further aspects, domain (b) of the first
polynucleotide (1) and/or domain (e) of the second polynucleotide (2) are
at least 2, at least 3, at least 4, at least 5, at least 6, at least 7,
at least 8, at least 9, at least 10, at least 11, at least 12, at least
13, at least 14, at least 15, at least 16, at least 17, at least 18, at
least 19, at least 20, at least 21, at least 22, at least 23, at least 24
nucleotides, at least 25, at least about 30, at least about 35, at least
about 40, at least about 45, at least about 50, at least about 60, at
least about 70, at least about 80, at least about 90, at least about 100,
at least about 120, at least about 140, at least about 160, at least
about 180, at least about 200, at least about 220, at least about 240, at
least about 260, at least about 280, at least about 300, at least about
320, at least about 340, at least about 360, at least about 380, at least
about 400, at least about 420, at least about 440, at least about 460, at
least about 480, at least about 500 or more nucleotides in length.

[0152] In some embodiments, compositions and methods described herein
include a second set of polynucleotides with the characteristics
described above for first and second polynucleotides. In some
embodiments, a plurality of sets is contemplated. These additional sets
of first and second polynucleotides can have any of the characteristics
described for first and second polynucleotides.

[0153] The rigid frame blocker polynucleotides are contemplated in one
aspect to comprise at least 30 nucleotides. In other aspects, the rigid
frame blocker polynucleotides can comprise at least 31 nucleotides, or at
least 32 nucleotides, or at least 33 nucleotides, or at least 34
nucleotides, or at least 35 nucleotides, or at least 36 nucleotides, or
at least 37 nucleotides, or at least 38 nucleotides, or at least 39
nucleotides, or at least 40 nucleotides, or at least about 45
nucleotides, or at least about 50 nucleotides, or at least about 55
nucleotides, or at least about 60 nucleotides, or at least about 65
nucleotides, or at least about 70 nucleotides, or at least about 75
nucleotides, or at least about 80 nucleotides, or at least about 85
nucleotides, or at least about 90 nucleotides, or at least about 95
nucleotides, or at least about 100 nucleotides, or at least about 105
nucleotides, or at least about 110 nucleotides, or at least about 150
nucleotides, or at least about 200 nucleotides, or at least about 250
nucleotides, or at least about 300 nucleotides, or at least about 350
nucleotides, or at least about 400 nucleotides, or at least about 450
nucleotides, or at least 500 nucleotides or more.

[0154] In some embodiments, the probe polynucleotide is from about 5
nucleotides in length to about 100 bases in length. In various aspects,
the probe polynucleotide comprises at least 5 nucleotides, or at least 6
nucleotides, or at least 7 nucleotides, or at least 8 nucleotides, or at
least 9 nucleotides, or at least 10 nucleotides, or at least 11
nucleotides, or at least 12 nucleotides, or at least 13 nucleotides, or
at least 14 nucleotides, or at least 15 nucleotides, or at least 16
nucleotides, or at least 17 nucleotides, or at least 18 nucleotides, or
at least 19 nucleotides, or at least 20 nucleotides, or at least 21
nucleotides, or at least 22 nucleotides, or at least 23 nucleotides, or
at least 24 nucleotides, or at least 25 nucleotides, or at least 26
nucleotides, or at least 27 nucleotides, or at least 28 nucleotides, or
at least 29 nucleotides, or at least 30 nucleotides, or at least 31
nucleotides, or at least 32 nucleotides, or at least 33 nucleotides, or
at least 34 nucleotides, or at least 35 nucleotides, or at least 36
nucleotides, or at least 37 nucleotides, or at least 38 nucleotides, or
at least 39 nucleotides, or at least 40 nucleotides, or at least about 45
nucleotides, or at least about 50 nucleotides, or at least about 55
nucleotides, or at least about 60 nucleotides, or at least about 65
nucleotides, or at least about 70 nucleotides, or at least about 75
nucleotides, or at least about 80 nucleotides, or at least about 85
nucleotides, or at least about 90 nucleotides, or at least about 95
nucleotides, or at least about 100 nucleotides of a DNA sequence that is
sufficiently complementary to a target polynucleotide region so as to
allow hybridization under appropriate conditions.

[0155] In some embodiments, the blocker polynucleotide is from about 5
nucleotides in length to about 1000 bases in length. In various aspects,
the blocker polynucleotide comprises at least 5 nucleotides, or at least
6 nucleotides, or at least 7 nucleotides, or at least 8 nucleotides, or
at least 9 nucleotides, or at least 10 nucleotides, or at least 11
nucleotides, or at least 12 nucleotides, or at least 13 nucleotides, or
at least 14 nucleotides, or at least 15 nucleotides, or at least 16
nucleotides, or at least 17 nucleotides, or at least 18 nucleotides, or
at least 19 nucleotides, or at least 20 nucleotides, or at least 21
nucleotides, or at least 22 nucleotides, or at least 23 nucleotides, or
at least 24 nucleotides, or at least 25 nucleotides, or at least 26
nucleotides, or at least 27 nucleotides, or at least 28 nucleotides, or
at least 29 nucleotides, or at least 30 nucleotides, or at least 31
nucleotides, or at least 32 nucleotides, or at least 33 nucleotides, or
at least 34 nucleotides, or at least 35 nucleotides, or at least 36
nucleotides, or at least 37 nucleotides, or at least 38 nucleotides, or
at least 39 nucleotides, or at least 40 nucleotides, or at least about 45
nucleotides, or at least about 50 nucleotides, or at least about 55
nucleotides, or at least about 60 nucleotides, or at least about 65
nucleotides, or at least about 70 nucleotides, or at least about 75
nucleotides, or at least about 80 nucleotides, or at least about 85
nucleotides, or at least about 90 nucleotides, or at least about 95
nucleotides, or at least about 100 nucleotides, or at least about 110
nucleotides, or at least about 120 nucleotides, or at least about 130
nucleotides, or at least about 140 nucleotides, or at least about 150
nucleotides, or at least about 160 nucleotides, or at least about 170
nucleotides, or at least about 180 nucleotides, or at least about 190
nucleotides, or at least about 200 nucleotides, or at least about 250
nucleotides, or at least about 300 nucleotides, or at least about 350
nucleotides, or at least about 400 nucleotides, or at least about 450
nucleotides, or at least about 500 nucleotides, or at least about 550
nucleotides, or at least about 600 nucleotides, or at least about 650
nucleotides, or at least about 700 nucleotides, or at least about 750
nucleotides, or at least about 800 nucleotides, or at least about 850
nucleotides, or at least about 900 nucleotides, or at least about 950
nucleotides, or at least about 1000 nucleotides of a polynucleotide
sequence that is sufficiently complementary to a target polynucleotide
region so as to allow hybridization under appropriate conditions. In
various embodiments, the blocker polynucleotide further comprises a
modified nucleotide, which in various aspects is an internal nucleotide
and/or is the nucleotide at its 5' end. In various embodiments, the
modified nucleotide is a locked nucleic acid. In some embodiments, the
blocker polynucleotide further comprises a blocking group at the 3' end
to prevent extension by a polymerase.

[0156] In some embodiments, the reverse primer polynucleotide is from
about 5 nucleotides in length to about 100 bases in length. In various
aspects, the reverse primer polynucleotide comprises at least 5
nucleotides, or at least 6 nucleotides, or at least 7 nucleotides, or at
least 8 nucleotides, or at least 9 nucleotides, or at least 10
nucleotides, or at least 11 nucleotides, or at least 12 nucleotides, or
at least 13 nucleotides, or at least 14 nucleotides, or at least 15
nucleotides, or at least 16 nucleotides, or at least 17 nucleotides, or
at least 18 nucleotides, or at least 19 nucleotides, or at least 20
nucleotides, or at least 21 nucleotides, or at least 22 nucleotides, or
at least 23 nucleotides, or at least 24 nucleotides, or at least 25
nucleotides, or at least 26 nucleotides, or at least 27 nucleotides, or
at least 28 nucleotides, or at least 29 nucleotides, or at least 30
nucleotides, or at least 31 nucleotides, or at least 32 nucleotides, or
at least 33 nucleotides, or at least 34 nucleotides, or at least 35
nucleotides, or at least 36 nucleotides, or at least 37 nucleotides, or
at least 38 nucleotides, or at least 39 nucleotides, or at least 40
nucleotides, or at least about 45 nucleotides, or at least about 50
nucleotides, or at least about 55 nucleotides, or at least about 60
nucleotides, or at least about 65 nucleotides, or at least about 70
nucleotides, or at least about 75 nucleotides, or at least about 80
nucleotides, or at least about 85 nucleotides, or at least about 90
nucleotides, or at least about 95 nucleotides, or at least about 100
nucleotides of a polynucleotide sequence that is sufficiently
complementary to a region of a polymerase-extended first polynucleotide
so as to allow hybridization under appropriate conditions. In some
embodiments, when the target polynucleotide is a double-stranded
polynucleotide, the reverse primer is complementary to a complementary
strand of the target polynucleotide. In some embodiments, the reverse
primer is a combination of first and second polynucleotides, as defined
herein.

V. Polynucleotide Base Structure

[0157] In some embodiments, the first polynucleotide is comprised of DNA,
modified DNA, RNA, modified RNA, PNA, or combinations thereof. In other
embodiments, the second polynucleotide is comprised of DNA, modified DNA,
RNA, modified RNA, PNA, or combinations thereof.

Vi. Polynucleotide Structure--Replication/Extension Blockers

[0158] An internal "replication blocker" is incorporated as needed when
termination of polymerase extension of a polynucleotide is desirable.
Similarly, 3' terminal extension blockers are also contemplated by the
disclosure. An "extension blocking group" or "extension blocker" is a
moiety that prevents initiation of extension from the 3' end of a
polynucleotide. For example, the second domain (c) of the first
polynucleotide (1), in another aspect, further comprises an extension
blocker at the 3' end of the second domain (c) to prevent extension by an
enzyme that is capable of synthesizing a nucleic acid. In further
aspects, the blocker polynucleotide comprises an extension blocker at its
3' end. In additional aspects, domain (g), which is positioned between
domain (d) and domain (e) in the second polynucleotide, comprises a
replication blocker. In still further aspects, the second polynucleotide
further comprises a fourth domain (h) positioned between domain (f) and
domain (e), wherein domain (h) comprises a replication blocker.

[0159] Terminal 3' extension blocking groups useful in the practice of the
methods include but are not limited to a 3' phosphate group, a 3' amino
group, a dideoxy nucleotide, and inverted deoxythymidine (dT). Internal
replication blocking groups useful in the practice of the methods include
but are not limited to an abasic site(s), a modified base(s), or a six
carbon glycol spacer (and in one aspect the six carbon glycol spacer is
hexanediol).

VII. Polynucleotide Structure--Complementarity

[0160] In some aspects, the second domain (d) of the second polynucleotide
(2) is at least about 70% complementary to the second domain (c) of the
first polynucleotide (1). In related aspects, the second domain (d) of
the second polynucleotide (2) is at least about 75%, at least about 80%,
at least about 85%, at least about 90%, at least about 95%, or about 100%
complementary to the second domain (c) of the first polynucleotide (1).

[0161] In another aspect, the blocker polynucleotide is at least about
75%, at least about 80%, at least about 85%, at least about 90%, at least
about 95%, or about 100% complementary to a sequence in the target
polynucleotide, and in yet another aspect, the probe polynucleotide is at
least about 75%, at least about 80%, at least about 85%, at least about
90%, at least about 95%, or about 100% complementary to a sequence in the
target polynucleotide, and/or a sequence in the blocker polynucleotide.

VIII. Hybridization Conditions

[0162] In some embodiments, the first (1) and second polynucleotide (2)
hybridize to each other under stringent conditions in the absence of a
template polynucleotide. In some embodiments, the first (1) and second
polynucleotides (2) do not hybridize to each other under stringent
conditions in the absence of a template polynucleotide. "Stringent
conditions" as used herein can be determined empirically by the worker of
ordinary skill in the art and will vary based on, e.g., the length of the
primer, complementarity of the primer, concentration of the primer, the
salt concentration (i.e., ionic strength) in the hybridization buffer,
the temperature at which the hybridization is carried out, length of time
that hybridization is carried out, and presence of factors that affect
surface charge of the polynucleotides. In general, stringent conditions
are those in which the polynucleotide is able to bind to its
complementary sequence preferentially and with higher affinity relative
to any other region on the target. Exemplary stringent conditions for
hybridization to its complement of a polynucleotide sequence having 20
bases include without limitation about 50% G+C content, 50 mM salt
(Na.sup.+), and an annealing temperature of 60° C. For a longer
sequence, specific hybridization is achieved at higher temperature. In
general, stringent conditions are such that annealing is carried out
about 5° C. below the melting temperature of the polynucleotide.
The "melting temperature" is the temperature at which 50% of
polynucleotides that are complementary to a target polynucleotide in
equilibrium at definite ion strength, pH and polynucleotide
concentration.

IX. Methods of Use

A. PCR

[0163] In target polynucleotide amplification methods described herein, a
third polynucleotide (3) and a fourth polynucleotide (4) are contemplated
for use in combination with the polynucleotide combination described
above, the third polynucleotide (3) comprising a first domain (a) that is
complementary to a complementary strand of the target polynucleotide
[relative to the strand to which the first domain of the first
polynucleotide (1) is complementary] at a first target complement
polynucleotide region and a second domain (c) comprising a unique
polynucleotide sequence, and the fourth polynucleotide (4) comprising a
first domain (f) that is complementary to the complementary strand of the
target polynucleotide [relative to the strand to which the second
polynucleotide is complementary] at a second complement target
polynucleotide region and a second domain (d) comprising a polynucleotide
sequence sufficiently complementary to the second domain (c) of the third
polynucleotide (3) such that the second domain (c) of the third
polynucleotide (3) and the second domain (d) of the fourth polynucleotide
(4) will hybridize under appropriate conditions. In some of these
aspects, the method further comprises contacting the target
polynucleotide and a complement of the target polynucleotide with the
first polynucleotide (1) and second polynucleotide (2) and the third
polynucleotide (3) and fourth polynucleotide (4) under conditions
sufficient to allow hybridization of the first domain (a) of the first
polynucleotide (1) to the first target polynucleotide region (A) of the
target polynucleotide, the first domain (f) of the second polynucleotide
(2) to the second target polynucleotide region (F) of the target
polynucleotide, the first domain (a) of the third polynucleotide (3) to
the first target domain of the complementary strand of the target
polynucleotide and the first domain (f) of the fourth polynucleotide (4)
to the second complement target polynucleotide region and extending the
first domains (i.e., priming domains) of the second and fourth
polynucleotides with a DNA polymerase under conditions which permit
extension of the second polynucleotide and the fourth polynucleotide. In
various embodiments, the third polynucleotide further comprises a third
domain (b) comprising a polymer sequence that is not sufficiently
complementary to hybridize to a domain in the first polynucleotide (1), a
domain in the second polynucleotide (2), a domain in the third
polynucleotide (3), a domain in the fourth polynucleotide (4) or a region
in the target polynucleotide, wherein domains in the third polynucleotide
are arranged 5'-a-b-c-3'. In related embodiments, the fourth
polynucleotide further comprises a third domain (e) comprising a
polynucleotide sequence that is not sufficiently complementary to
hybridize to a domain in the first polynucleotide (1), a domain in the
second polynucleotide (2), a domain in the third polynucleotide (3), a
domain in the fourth polynucleotide (4) or a region in the target
polynucleotide, wherein domains in the second polymer are arranged
5'-d-e-f-3', wherein under conditions in which region (A) in the target
polynucleotide specifically hybridizes to domain (a) and region (F) of
the target polynucleotide specifically hybridizes to domain (f), domain
(c) of the third polynucleotide (3) hybridizes to domain (d) of the
fourth polynucleotide (4) and neither domain (b) nor domain (e)
hybridizes to a domain in the first polynucleotide (1), a domain in
second polynucleotide (2), a domain in the third polynucleotide (3), a
domain in the fourth polynucleotide (4) or a region in the target
polynucleotide.

[0164] In some aspects, an extension blocker as described herein above is
attached to the first polynucleotide (1) and/or the third polynucleotide
(3) at their 3' ends which blocks extension by an enzyme that is capable
of synthesizing a nucleic acid. Blocking groups useful in the practice of
the methods include but are not limited to a 3' phosphate group, a 3'
amino group, a dideoxy nucleotide, and inverted deoxythymidine (dT).

[0165] In various embodiments, the target polynucleotide, the complement
of the target polynucleotide or both has a secondary structure that is
denatured by hybridization of the first domain (a) of the first
polynucleotide and/or the first domain (a) of the third polynucleotide to
a target polynucleotide.

[0166] One of ordinary skill in the art will recognize that the
polynucleotides of combinations of the present disclosure can be used to
prime either one or both ends of a given PCR amplicon. As used herein, an
"amplicon" is understood to mean a portion of a polynucleotide that has
been synthesized using amplification techniques. It is contemplated that
any of the methods of the present disclosure that comprise more than one
polynucleotide combination may utilize any combination of standard primer
and polynucleotide combination, provided at least one of the primers is a
polynucleotide combination as described herein.

[0167] In various embodiments and as described herein, a polynucleotide
primer combination of the disclosure is used to detect a mutation in a
target polynucleotide versus a non-target polynucleotide. Accordingly, in
some embodiments a polynucleotide primer combination is used to detect a
polymorphism in a target polynucleotide (see FIG. 3). In FIG. 3, the
polynucleotide sequence of domain (f) specifically hybridizes to a mutant
DNA sequence (region (F)), but comprises at least one mismatch with a
wildtype DNA sequence (region (F*)). Thus, region (f) can specifically
hybridize to region (F) and serve to prime an extension product from the
mutant DNA target polynucleotide.

[0168] In related embodiments, a polynucleotide primer combination of the
disclosure that is used to detect a mutation in a target polynucleotide
versus a non-target polynucleotide further comprises a blocker
polynucleotide (FIG. 4). FIG. 4 depicts target polynucleotide
discrimination with a polynucleotide primer combination of the disclosure
and a blocker polynucleotide. In some aspects, and as shown in FIG. 4,
the blocker polynucleotide is degraded following displacement from the
target polynucleotide. Specifically, in the presence of a target
polynucleotide, mutation-specific domain (f) first invades region (F) and
displaces the imperfectly annealed 5' tail of the blocker polynucleotide.
Then DNA polymerase with 5' exonuclease activity (for example and without
limitation, wildtype Tag DNA polymerase) cleaves the 5' flap of the
blocker polynucleotide. At the next step DNA polymerase replicates the
DNA template in a nick-translation mode until the blocking
oligonucleotide becomes too short to maintain a stable interaction with
DNA and dissociates from the template allowing polymerase to proceed in
normal replication mode. In the case of wildtype DNA (FIG. 4),
mutation-specific domain (f) is unable to invade and displace the
perfectly annealed blocker polynucleotide, and thus cannot prime DNA
synthesis.

[0169] In some embodiments, a primer extension reaction and polymerase
chain reactions as depicted in FIGS. 6-9 with one or two polynucleotide
primer combinations of the disclosure are shown. Reactions depicted in
FIGS. 6-9 are, in various aspects, executed using real-time PCR
instruments (quantitative PCR) in the presence of staining dyes, which
for example and without limitation include SybrGreen, or in the presence
of probes, for example and without limitation TaqMan, beacon and
Scorpions. In some aspects, the reactions use mutation-specific
polynucleotide primers of the disclosure as described in FIG. 3. In
further aspects, the reactions use blocker polynucleotides as described
in FIG. 4.

[0170] Also provided by the disclosure are embodiments wherein a
polynucleotide primer combination of the disclosure further comprises a
replication blocking domain (g) (FIG. 10). In this case the
polynucleotide primer combination has an additional element, specifically
a replication blocking domain (g) located between domains (d) and (e) of
the second polynucleotide (2). Domain (g) can, in various aspects, be a
modified base or linker or a polymer that would not allow a DNA
polymerase to replicate domain (d) after finishing replication of domain
(e) but rather would terminate replication at domain (g). In some
aspects, the purpose of domain (g) is to eliminate sequence (c) of the
first polynucleotide (1) from the 3' end of the PCR product.

[0171] In additional embodiments, a polymerase chain reaction with two
polynucleotide primer combinations of the disclosure, each comprising a
replication-blocking domain (g) and two universal primers (e2 and
e4 in FIGS. 11 and 12) that have no complementarity to the target
polynucleotide. In some aspects, all four primers are present in the
reaction tube from the start. During the first four steps, the first
polynucleotide (1), second polynucleotide (2), third polynucleotide (3)
and fourth polynucleotide (4) form a PCR amplicon. During the next PCR
cycles, amplification can be supported by either the second
polynucleotide (2) and the fourth polynucleotide (4) or by universal
primers e2 and e4, or by all 4 primers (FIG. 12). If the
concentration of the second polynucleotide (2) and the fourth
polynucleotide (4) is substantially lower than the concentration of
universal primers e2 and e4, or primers e2 and e4
have a higher stability than the second polynucleotide (2) and the fourth
polynucleotide (4) due to introduced base modifications (for example and
without limitation, LNAs) the amplification will be mostly accomplished
by universal primers e2 and e4.

[0172] In a further embodiment, a polynucleotide primer combination of the
disclosure is provided in which region (A) is 5' to region (F) in the
target polynucleotide. This polynucleotide primer combination is, in some
aspects, contemplated for use in detecting a mutation in a target
polynucleotide versus a non-target polynucleotide (FIG. 13). Due to the
flexibility of domain (e), the interaction of short domain (f) with
target polynucleotide region (F) is highly sensitive to any defect within
region (F). In the absence of mismatches within the short duplex between
domain (f) and region (F), the formed priming complex (see FIG. 13)
initiates extension of a product polynucleotide from the 3' end of domain
(f). If region (F) has a mutation, and corresponding mutation-specific
domain (f) of the second polynucleotide (2) forms a stable duplex with
mutated region (F), the formed primer complex can initiate extension of a
product polynucleotide and degradation and/or displacement of domain (a)
from the 3' end of the mutation-specific domain (f). If region (F) has no
mutation (i.e., is a wildtype sequence (F*)), the mutation-specific
domain (f) of the second polynucleotide (2) cannot form a stable duplex
with region (F*) and cannot initiate extension of a product
polynucleotide and degradation and/or displacement of domain (a) from the
3' end of the mutation-specific domain (f) (FIG. 13).

[0173] In another embodiment, a polynucleotide primer combination of the
disclosure is provided in which region (A) is 5' to region (F) in a
target polynucleotide, it is further contemplated that at least a portion
of domain (a) acts as a blocker polynucleotide (FIG. 14). Use of a
blocker polynucleotide enables the detection of a few or even single
mutant target polynucleotides in the presence of a very large number of
non-target polynucleotides, for example and without limitation 1 target
polynucleotide: 104 non-target polynucleotides, or 1 target
polynucleotide: 105 non-target polynucleotides. Ratios of target to
non-target polynucleotides that can be detected by polynucleotide primer
combinations of the disclosure are from at least about 1:10 to at least
about 1:1010.

[0174] In some embodiments, the length of domain (a) of the first
polynucleotide (1) is extended in the 5' direction, allowing domain (a)
of the first polynucleotide (1) to overlap with domain (f) of the second
polynucleotide (2). In these aspects, domain (a) serves as a blocker
oligonucleotide that increases the specificity of mutation selection
during primer extension and PCR.

[0175] The blocking portion (a5') of domain (a) is in various aspects
designed to be complementary to a region (F*) in a non-target
polynucleotide and include a mismatch with a region (F) in a target
polynucleotide. The blocking portion (a5') of domain (a) interacts
with region (F*) or a part of region (F*) that comprises a variant base,
thus overlapping completely or partially with domain (f) of the second
polynucleotide (2). Blocking portion (a5') of domain (a), in various
aspects, comprises a modified base, including but not limited to LNAs and
PNAs.

[0176] As shown in FIG. 14, with respect to a target polynucleotide,
mutation-specific domain (f) first invades region (F) and displaces the
imperfectly annealed 5' tail of domain (a) of the first polynucleotide
(1). Then a DNA polymerase with 5' to 3' exonuclease activity (for
example and without limitation, Tag DNA polymerase) cleaves the displaced
region (a5') of domain (a) of the first polynucleotide (1). Next,
the polymerase replicates the polynucleotide template in a
nick-translation mode until domain (a) of the first polynucleotide (1)
becomes too short to maintain a stable interaction with the target
polynucleotide and dissociates from the template allowing the polymerase
to proceed in replication mode. In the case of a non-target
polynucleotide, domain (f) is unable to invade and displace the perfectly
annealed domain (a) of the first polynucleotide (1), and thus cannot
prime polynucleotide extension. Accordingly, use of domain (a) with a
blocking function results in improved discrimination of a rare target
polynucleotide in the presence of abundant non-target polynucleotides
using a polynucleotide primer combination as described above.

[0177] In further embodiments, a polynucleotide primer combination is
provided wherein domain (a) further comprises a detectable marker and a
quencher that quenches the detectable marker. As shown in FIG. 15,
incorporation of a detectable marker and quencher into binding domain (a)
adds a domain (a) 5'-detection probe function to a polynucleotide primer
combination. Detectable marker and quencher molecules can be positioned
as shown in FIG. 15, or in some aspects can be positioned in the reverse
orientation (i.e., detectable marker can be positioned 3' of the
quencher). A short distance (for example and without limitation, at least
1, at least 2, at least 3, at least 4, at least 5, at least 6, at least
7, at least 8, at least 9, at least 10, at least 11, at least 12, at
least 13, at least 14, at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, at least 25, at least 26, at least 27, at least 28, at least
29, at least 30 or more nucleotides) between the detectable marker and
quencher within domain (a) would result in quenching of the detectable
marker and absence of detection. Upon primer extension and degradation of
domain (a) the detectable marker or the quencher will be released into
solution and generate detectable signal. Extension of domain (f) would
result in a detectable signal generated from the target polynucleotide.
Lack of extension of domain (f) results in no detectable signal from the
non-target polynucleotide. For generation of a detectable signal by
primer combinations containing detectable marker and quencher molecules
within domain (a), it is important for the polymerase used for extension
or amplification to have intrinsic 5' to 3' exonuclease activity.
Examples of thermophilic DNA polymerases with 5' to 3' exonuclease
activity include but are not limited to Taq DNA polymerase, Tth DNA
polymerase, family of DyNAzyme DNA polymerases from Termus brockianus,
Bst DNA polymerase, holoenzyme; a non-limiting example of a mesophilic
DNA polymerase with 5' to 3' exonuclease activity is E. coli DNA
polymerase I.

[0178] In some embodiments, a polynucleotide primer combination is
provided wherein domain (a) further comprises a detectable marker and a
quencher that quenches the detectable marker, and wherein domain (a)
further comprises a blocker polynucleotide domain (a5') as described
above, and depicted in FIG. 16.

[0179] A polynucleotide primer combination is also provided, in certain
embodiments, that further comprises a replication blocker domain within
the second polynucleotide (2) (see FIG. 17). A polynucleotide primer
combination can thus have an additional element, specifically a
replication blocker domain, located either between domains (d) and (e)
(i.e., domain (g)), or between domains (f) and (e) (i.e., domain (h)).
The replication blocker domain is, in various aspects, an abasic site, a
modified base or linker, or a polymer that does not allow DNA polymerase
to replicate beyond but rather terminates replication at the position of
the (g) or (h) domain. The purpose of replication blocker domain is to
prevent DNA polymerase from replicating domain (d) (in the case of domain
(g)), or from replicating domains (e) and (d) (in the case of domain
(h)). Use of a polynucleotide primer combination without a replication
blocker domain is depicted in FIG. 18. Polynucleotide primer combinations
of the disclosure can be used for quantitative PCR, mutation-specific
PCR, mutation-specific PCR enhanced by an overlapping blocking domain,
and for mutation-specific PCR with combined blocking domain and detection
probe function. During the first 6 steps of the PCR process described in
FIG. 18, two polynucleotide primer combinations (forward and reverse)
facilitate formation of the PCR amplicon. After formation of the PCR
amplicon, further amplification can be supported by polynucleotide primer
combinations 2 and 4 without absolute necessity of first and third
polynucleotides (1) and (3) (although in some aspects they can still
participate as fluorescent probes or blockers, and help to generate a
specific signal).

[0180] In embodiments wherein the second polynucleotide (2) comprises a
replication blocker domain (g), FIGS. 19 and 20 depict their use in PCR.
A polynucleotide primer combination with a (g) domain replication blocker
can be used for quantitative PCR, mutation-specific PCR,
mutation-specific PCR enhanced by an overlapping blocking domain, and for
mutation-specific PCR with the combined blocking and detection probe
function. During the first 6 steps of the PCR process described in FIG.
20, two primers (forward and reverse) facilitate formation of the PCR
amplicon. After formation of the PCR amplicon, further amplification can
be supported by universal primers e2 and e4.

[0181] The disclosure also provides a polynucleotide primer combination
wherein the second polynucleotide (2) further comprises a replication
blocker domain (h), wherein domain (h) is positioned between domains (e)
and (f) (FIG. 21). The potential for a polynucleotide primer combination
of the disclosure to form a primer-dimer at low temperature cannot be
amplified at a higher temperature due to the presence of the replication
blocker domain (h).

[0182] In embodiments wherein the second polynucleotide (2) comprises a
replication blocker domain (h), FIGS. 22 and 23 A-G depict their use in
PCR. In the case of polynucleotide primer combinations without a
replication blocking domain or with a (g) domain, fully-assembled
polynucleotide primer combinations are only necessary for the first few
cycles of the PCR process. Later PCR amplification can be supported by
the second polynucleotide (2) alone or by primers corresponding to their
internal (e) domain sequences. Polynucleotide primer combinations with an
(h) domain function differently. They cannot support PCR without the
presence of the first and third polynucleotides (1) and (3) during the
whole process due to the very short size of their functional priming
domain. For this reason, any primer-dimers formed by a polynucleotide
primer combination that contains an (h) domain cannot be amplified at
temperatures typical for a standard PCR reaction (>50° C.). For
example and without limitation, a polynucleotide primer combination with
an (h) domain can be used for quantitative PCR, mutation-specific PCR,
mutation-specific PCR enhanced by an overlapping blocking domain, and for
mutation-specific PCR with combined blocking and detection probe domains.

[0183] In some embodiments, a polynucleotide primer combination is
provided wherein the first polynucleotide (1) comprises a self-blocking
domain (FIG. 24). A polynucleotide primer combination with a
self-blocking domain represents a case where the first polynucleotide (1)
plays a dual function: as a stable binding domain (a) and as a
competitive blocker domain for the target polynucleotide specific domain
(f) of the second polynucleotide (2). To achieve this goal the
polynucleotide primer combination is designed such that the binding
domain (a) of the first polynucleotide (1) has two adjacent domains:
binding domain (a) and binding-blocking domain (j) that are fully
complementary to two adjacent target regions (A) and (F*) of the
non-target polynucleotide, and not fully complementary to the (F) region
of the target polynucleotide. As a result, the first polynucleotide (1)
will form a stable duplex between domains (a) and (j) and (A) and (F*) of
the non-target polynucleotide and a destabilized duplex between domain
(f) and region (F) of the target polynucleotide. Formation of a stable
complex between the first polynucleotide (1) and the non-target
polynucleotide within regions (A) and (F*) will outcompete formation of a
less stable duplex between the priming domain (f) of the second
polynucleotide (2) and the non-target polynucleotide, thus preventing
extension of domain (f) on the non-target polynucleotide. Formation of an
unstable duplex between domain (j) of the first polynucleotide (1) and
the mismatched region (F) of the target polynucleotide will promote
competition between domain (f) of the second polynucleotide (2) and
domain (j) of the first polynucleotide (1). This results in formation of
a stable (f)/(F) duplex and extension of domain (f) on the target
polynucleotide. As a result, self-blocking polynucleotide primer
combinations provide greater specificity for amplification and detection
of target alleles that are present at very low copy number in the
presence of a large number of non-target alleles.

[0184] In a related embodiment, a polynucleotide primer combination is
provided wherein the first polynucleotide (1) comprises non-contiguous
domains (a) and (j) (FIG. 25). The situation presented in FIG. 25 differs
from that described in FIG. 24 only by the fact that the two target
regions (A) and (F) are not adjacent to one another but are separated by
region (X). To adjust to this difference, the self-blocking first
polynucleotide (1) depicted in FIG. 25 has a first polynucleotide (1)
portion with three domains: domains (a) and (j) that are fully
complementary to regions (A) and (F*) of the non-target polynucleotide,
and domain (p) that represents a unique non-complementary spacer between
domains (a) and (j). The self-blocking polynucleotide primer combination
described in FIG. 25 functions similarly to the primer combination
described in FIG. 24.

B. Simple Second Strand Synthesis

[0185] In another embodiment, a method of amplifying a target
polynucleotide is provided using the first (1) and second polynucleotides
(2) comprising contacting the target polynucleotide with the first (1)
and second polynucleotides (2) disclosed herein under conditions
sufficient to allow hybridization of the first domain (a) of the first
polynucleotide (1) to the first target polynucleotide region (A) of the
target polynucleotide and the first domain (f) of the second
polynucleotide (2) to the second target polynucleotide region (F) of the
target polynucleotide, and extending the first domain (f) (i.e., priming
domain) of the second polynucleotide with a DNA polymerase under
conditions which permit extension of the first domain of the first
polynucleotide. In some aspects, the second polynucleotide (2) (with
associated polynucleotide product extended therefrom) and first
polynucleotide are then denatured from the target polynucleotide and
another set of first and second polynucleotides are allowed to hybridize
to a target polynucleotide.

[0186] In one aspect, the first polynucleotide (1) and the second
polynucleotide (2) hybridize sequentially to the target polynucleotide.
In another aspect, the first domain (a) of the first polynucleotide (1)
hybridizes to the target before the first domain (f) of the second
polynucleotide (2) hybridizes to the target polynucleotide. In yet
another aspect, the first domain (f) of the second polynucleotide (2)
hybridizes to the target polynucleotide before the first domain (a) of
the first polynucleotide (1) hybridizes to the target polynucleotide. In
another aspect, the first domain (a) of the first polynucleotide (1) and
the first domain (f) of the second polynucleotide (2) hybridize to the
target polynucleotide concurrently.

[0187] In various embodiments, the target polynucleotide includes but is
not limited to chromosomal DNA, genomic DNA, plasmid DNA, cDNA, RNA, a
synthetic polynucleotide, a single stranded polynucleotide, or a double
stranded polynucleotide. In one aspect, the target is a double stranded
polynucleotide and the first domain (a) of the first polynucleotide (1)
and the first domain (f) of the second polynucleotide (2) hybridize to
the same strand of the double stranded target polynucleotide. In another
aspect, the second domain (c) of the first polynucleotide (1) and the
second domain (d) of the second polynucleotide (2) hybridize prior to
hybridization of the first polynucleotide (1) and the second
polynucleotide (2) to the target polynucleotide.

[0188] In an embodiment, the first polynucleotide (1) and the second
polynucleotide (2) hybridize to the target polynucleotide concurrently
and the third polynucleotide (3) and the fourth polynucleotide (4)
hybridize to the complement of the target polynucleotide concurrently,
the first polynucleotide (1) and the second polynucleotide (2)
hybridizing to the target polynucleotide at the same time that the third
polynucleotide (3) and the fourth polynucleotide (4) hybridize to the
complement of the target polynucleotide.

[0189] In another embodiment, the first polynucleotide (1), the second
polynucleotide (2), the third polynucleotide (3) and the fourth
polynucleotide (4) do not hybridize to the target polynucleotide and the
complement of the target polynucleotide at the same time.

[0190] In yet another embodiment, the second domain (c) of the first
polynucleotide (1) and the second domain (d) of the second polynucleotide
(d) hybridize prior to hybridizing to the target polynucleotide. In
another embodiment, the second domain (c) of the third polynucleotide (3)
and the second domain (d) of the fourth polynucleotide (4) hybridize
prior to hybridizing to the complement of the target polynucleotide.

[0191] In an embodiment, the second domain (c) of the first polynucleotide
(1) and the second domain (d) of the second polynucleotide (2) hybridize
prior to hybridizing to the target polynucleotide and the second domain
(c) of the third polynucleotide (3) and the second domain (d) of the
fourth polynucleotide (4) hybridize prior to hybridizing to the
complement of the target polynucleotide.

[0192] In another embodiment, the target polynucleotide contains a
mutation in the region to which the first domain (f) of the second
polynucleotide hybridizes to the target polynucleotide. In some
embodiments, the target polynucleotide is fully complementary in the
region to which the first domain (f) of the second polynucleotide (2)
hybridizes to the target polynucleotide. In some embodiments, the
non-target polynucleotide is not fully complementary in the region to
which the first domain (f) of the second polynucleotide (2) hybridizes to
the non-target polynucleotide. In another embodiment, the target
polynucleotide contains a mutation in the region to which the first
domain (f) of the fourth polynucleotide (4) hybridizes to the target
polynucleotide. In some aspects, the mutation is a destabilizing
mutation, an insertion, a deletion, a substitution or an inversion. In
related aspects, the destabilizing mutation prevents extension of the
second polynucleotide (2), or the fourth polynucleotide (4), or both.

C. Multiplexing

[0193] In an embodiment, the extension by an enzyme that is capable of
synthesizing a nucleic acid is a multiplex extension, the first domain
(f) of the second polynucleotide having the property of hybridizing to
more than one region in the target polynucleotide. In a related
embodiment, the extension by an enzyme that is capable of synthesizing a
nucleic acid is a multiplex extension, the first domain (f) of the fourth
polynucleotide (4) having the property of hybridizing to more than one
locus in the target polynucleotide.

[0194] In related embodiments, multiplex PCR is performed using at least
two polynucleotide primers to amplify more than one polynucleotide
product. In some aspects of these embodiments, each polynucleotide primer
used for multiplex PCR is a polynucleotide combination as disclosed
herein. In other aspects, at least one polynucleotide primer used for
multiplex PCR is a polynucleotide combination as disclosed herein.

[0195] In another embodiment, multiplex PCR is performed using multiple
fixer polynucleotides and are directed against genomic repeated
sequences. In another embodiment, the fixer polynucleotides are comprised
of random sequences. In some of these aspects, multiple fixer
polynucleotides refers to about 10 polynucleotide sequences. In other
aspects, multiple fixer polynucleotides refers to about 15, about 20,
about 25, about 30, about 35, about 40, about 45, about 50, about 55,
about 60, about 65, about 70, about 75, about 80, about 85, about 90,
about 95, about 100, about 150, about 200, about 250, about 300, about
350, about 400, about 450, about 500, about 550, about 600, about 650,
about 700, about 750, about 800, about 850, about 900, about 950, about
1000 or more polynucleotide sequences. These fixer polynucleotide
sequences would provide a multitude of "fixed" locations in the genome to
which a multitude of primer polynucleotides could then bind, taking
advantage of the unique complementary polynucleotide sequences present in
both the primer and fixer polynucleotides as described herein.

[0196] Thus, in an embodiment, multiple target polynucleotide sequences
can be analyzed in a single reaction vessel. In some aspects, this
multiplex reaction is performed with a polynucleotide primer combination
of the disclosure that further comprises a replication blocker. In one
aspect, the replication blocker is domain (g) as described herein. FIG. 5
depicts three forward polynucleotide primer combinations of the
disclosure for the multiplexed PCR that have flexible domains (e2),
(e4) and (e6), each with the same universal sequence `e`. Three
reverse polynucleotide primer combinations of the disclosure that are
used for the multiplexed PCR are also depicted in FIG. 5, which have
flexible domains (e8), (e10) and (e12) each with the same
universal sequence `E` that is different from universal sequence `e`.
After the first three PCR cycles, all three targeted amplicons will be
terminated by two universal sequences `e` and `E`, so further
amplification of all amplicons can be supported by the two universal
primers e and E. These aspects involve three co-amplified targeted
regions, but a worker of ordinary skill in the art will appreciate that
this number can be substantially greater and involve 10, 100, 1,000,
10,000 or more targeted regions in one multiplexed PCR.

D. Real-Time PCR

[0197] Primer combinations of the disclosure are useful for real-time PCR.
Analysis and quantification of rare transcripts, detection of pathogens,
diagnostics of rare cancer cells with mutations, or low levels of
aberrant gene methylation in cancer patients are among the problems that
can be solved by improved real-time PCR assays that combine high
sensitivity and specificity of target amplification, high specificity of
target detection, the ability to selectively amplify and detect a small
number of cancer-specific mutant alleles or abnormally methylated
promoters in the presence of thousands of copies of normal DNA, analysis
and quantification of low copy number RNA transcripts, detection of
fluorescence traces the ability to multiplex 4-5 different targets in one
assay to maximally utilize capabilities of current real-time thermal
cyclers. A fluorophore is positioned at the 5' end of the second
polynucleotide (2), and a quencher is positioned at the 3' end of the
first polynucleotide (1). In this arrangement, no fluorescence is
detected when the first (1) and second (2) polynucleotides are hybridized
(since the fluorophore is positioned adjacent to the quencher). However,
following extension of the primer polynucleotide during PCR, the first
polynucleotide (1) and second polynucleotide (2) will become separated
during the denaturation phase of PCR, thus creating distance between the
fluorophore and the quencher and resulting in a detectable fluorescent
signal.

[0198] Primer combinations of the disclosure and a probe polynucleotide
can also be used for real-time PCR. The probe polynucleotide is labeled
with a fluorophore on its 5' end, a quencher on its 3' end, and in some
embodiments, an additional internal quencher. When the first
polynucleotide is extended by a polymerase with 5' to 3' exonuclease
activity, such as Tag polymerase, the label is cleaved and is no longer
quenched, resulting in increased signal from the label. In some
embodiments, the probe polynucleotide is a molecular beacon probe. In
short, a molecular beacon probe is comprised of a nucleotide sequence
with bases on its 5' and 3' ends that are complementary and form a
hairpin structure in the absence of a target polynucleotide. The
molecular beacon probe also comprises a quencher at its 3' end (or 5'
end) and a fluorescent label at its 5' end (or 3' end) such that there is
no detectable signal from the label when the target polynucleotide is not
present. The molecular beacon probe also comprises a sequence that is
complementary to the target polynucleotide such that, in the presence of
the target, hybridization of the probe to the target polynucleotide
causes the dissociation of the hairpin structure and loss of quenching,
resulting in a detectable fluorescent signal.

[0199] Primer combinations of the disclosure and a blocker polynucleotide
can also be used in combination for real-time PCR. The primer
polynucleotide (i.e., "second polynucleotide (2)") is labeled with a
fluorophore on its 5' end, and the fixer polynucleotide (i.e., "first
polynucleotide (1)") is labeled with a quencher on its 3' end. The
blocker polynucleotide is complementary to a target polynucleotide region
located immediately 5' of the second target polynucleotide region (F). In
some embodiments, the blocker polynucleotide overlaps with the first
domain (f) of the second polynucleotide (2). In other words, the
nucleotide(s) at the 3' end of the second polynucleotide (2) and the
nucleotide(s) at the 5' end of the blocker polynucleotide would be
complementary to the same nucleotide(s) of the target polynucleotide. In
related embodiments, the nucleotide(s) at the 3' end of the second
polynucleotide (2) and the nucleotide(s) at the 5' end of the blocker
polynucleotide are different. In these embodiments, the nucleotide(s) at
the 3' end of the second polynucleotide (2) would hybridize to the target
polynucleotide when it is complementary to the target polynucleotide at
the appropriate position(s), thus allowing for extension of the second
polynucleotide under the appropriate conditions. Following extension of
the primer polynucleotide during PCR, the primer polynucleotide and fixer
polynucleotide will become separated during the denaturation phase of
PCR, thus creating distance between the fluorophore and the quencher and
resulting in a detectable fluorescent signal. In related embodiments, the
nucleotide at the 5' end of the blocker polynucleotide would hybridize to
the non-target polynucleotide when it is complementary to the non-target
polynucleotide at the appropriate position, thus blocking extension of
the second polynucleotide. In this arrangement, no fluorescence is
detected when the primer and fixer polynucleotides are hybridized (since
the fluorophore is positioned adjacent to the quencher). In various
embodiments, the nucleotide at the 3' end of the blocker polynucleotide
is modified to prevent extension by a polymerase. This system allows for
detection of, for example and without limitation, single nucleotide
polymorphisms with great sensitivity and specificity.

[0200] Primer combinations of the disclosure, blocker polynucleotides, and
probe polynucleotides are also used in combination for real-time PCR. In
related embodiments, the second polynucleotide (2) used in this
combination comprises a modified nucleotide as the nucleotide at its 3'
end and the blocker polynucleotide comprises a modified nucleotide as the
nucleotide at its 5' end. In various aspects, the second polynucleotide
(2) and/or the blocker polynucleotide comprises at least one modified
nucleotide that is 1, 2, 3, 4, or 5 nucleotides from their 3' or 5' end,
respectively. In some embodiments, the modified nucleotide is a locked
nucleic acid.

[0201] In some aspects, the above embodiments further comprise a reverse
primer polynucleotide. The reverse primer is complementary to a region in
the polynucleotide created by extension of the second polynucleotide. As
is apparent, in some embodiments the reverse primer is also complementary
to the complementary strand of the target polynucleotide when the target
polynucleotide is one strand of a double-stranded polynucleotide.
Inclusion of a reverse primer allows for amplification of the target
polynucleotide. In various aspects, the reverse primer is a "simple"
primer wherein the sequence of the reverse primer is designed to be
sufficiently complementary over its entire length to hybridize to a
target sequence over the entire length of the primer. A simple primer of
this type is in one aspect, 100% complementary to a target sequence,
however, it will be appreciated that a simple primer with complementarity
of less than 100% is useful under certain circumstances and conditions.

[0202] In other aspects, a reverse primer is a separate polynucleotide
primer combination that specifically binds to regions in a sequence
produced by extension of a polynucleotide from the first domain (f) of
the second polynucleotide (2) in a primer pair combination used in a
first reaction.

[0203] In various aspects, the methods described herein provide a change
in sequence detection from a sample with a non-target polynucleotide
compared to sequence detection from a sample with a target
polynucleotide. In some aspects, the change is an increase in detection
of a target polynucleotide in a sample compared to sequence detection
from a sample with a non-target polynucleotide. In some aspects, the
change is a decrease in detection of a target polynucleotide in a sample
compared to sequence detection from a sample with a non-target
polynucleotide.

[0204] Due to the increased specificity of the polynucleotides described
herein, real-time PCR can be performed in the presence of SYBR green dye
to achieve a specificity that is equivalent to that achieved using
TaqMan, molecular beacon probes or Scorpion primers but at a greatly
reduced cost.

[0205] In one embodiment, the primer polynucleotide (i.e., "second
polynucleotide (2)") is labeled with a fluorescent molecule at its 5' end
and a second quenching polynucleotide (i.e., "universal quencher
polynucleotide") that is labeled at its 3' end with a quencher are both
hybridized to the second domain (c) of the fixer polynucleotide (i.e.,
"first polynucleotide (1)"), which comprises a blocking group at its 3'
end to prevent extension from a DNA polymerase. This complex has no
fluorescence in this state but will fluoresce when the complex is
displaced (denatured) following extension of the primer polynucleotide by
a DNA polymerase.

[0206] In another embodiment, the primer polynucleotide (i.e., "second
polynucleotide (2)") comprising a fluorophore at its 5' end is hybridized
to a fixer polynucleotide (i.e., "first polynucleotide (1)") comprising a
quencher at its 3' end. The complex has no fluorescence when hybridized,
but will fluoresce when the complex is displaced (denatured) following
extension of the primer polynucleotide by a DNA polymerase. In another
aspect of the method, multiplex real-time PCR is performed using two sets
of polynucleotide combinations, wherein one polynucleotide in each primer
set is labeled with a fluorophore, and the two fluorophores are
distinguishable from each other.

[0207] In another embodiment, the primer polynucleotide (i.e., "second
polynucleotide (2)") comprises a fluorophore, a quencher on its 3' end,
and these two labels are separated by a stretch of RNA or RNA/DNA
oligonucleotides (i.e., "probe polynucleotide"). In some aspects, the
probe polynucleotide further comprises an internal Zen quencher. In some
aspects, a fluorescent signal is generated upon creation and degradation
of the RNA/DNA hybrid by a thermostable RNase H and release of a free
fluorophore (or quencher) into solution.

[0208] In some embodiments, one fixer polynucleotide (i.e., "first
polynucleotide (1)") may be used in combination with 2, 3, 4, 5 or more
primer polynucleotides (i.e., "second polynucleotides (2)") for
simultaneous multiplex detection of several mutations in one real-time
PCR assay.

[0209] In another embodiment, a kit is provided comprising, e.g., a
package insert and any of the primer combinations of the disclosure,
which can in various aspects be fluorescently labeled).

E. Primer Extension

[0210] The primer compositions disclosed herein can be used in any method
requiring or utilizing primer extension. For example, primer extension
can be used to determine the start site of RNA transcription for a known
gene. This technique requires a labeled primer polynucleotide combination
as described herein (usually 20-50 nucleotides in length) which is
complementary to a region near the 3' end of the gene. The polynucleotide
combination is allowed to anneal to the RNA and reverse transcriptase is
used to synthesize complementary DNA (cDNA) to the RNA until it reaches
the 5' end of the RNA. By analyzing the product on a polyacrylamide gel,
it is possible to determine the transcriptional start site, as the length
of the sequence on the gel represents the distance from the start site to
the labeled primer.

[0212] Isothermal DNA amplification may be performed as taught in U.S.
Pat. No. 7,579,153 using the advanced polynucleotide technology described
herein. Briefly, isothermal DNA amplification comprises the following
steps: (i) providing a double stranded DNA having a hairpin at one end,
the polynucleotide at the other end, and disposed therebetween a promoter
sequence oriented so that synthesis by an RNA polymerase recognizing the
promoter sequence proceeds in the direction of the hairpin; (ii)
transcribing the double stranded DNA with an RNA polymerase that
recognizes the promoter sequence to form an RNA transcript comprising
copies of the promoter sequence and the polynucleotide; (iii) generating
a complementary DNA from the RNA transcript; (iv) displacing a 5' end of
the RNA transcript from the complementary DNA so that the hairpin is
reconstituted; and (v) extending the hairpin to generate the double
stranded DNA containing a reconstituted promoter sequence, the RNA
polymerase recognizing the reconstituted promoter sequence and
synthesizing RNA transcripts. In a preferred embodiment, the step of
generating includes forming a heteroduplex of said complementary DNA and
said RNA transcript and wherein said step of displacing includes treating
the heteroduplex with a helicase.

G. Fluorescence In Situ Hybridization (FISH)

[0213] The advanced polynucleotide technology described herein can also be
used to practice FISH. FISH is a cytogenetic technique used to detect and
localize the presence or absence of specific DNA sequences on
chromosomes. FISH uses fluorescent probes that bind to only those parts
of the chromosome with which they show a high degree of sequence
similarity. Fluorescence microscopy can be used to find out where the
fluorescent probe bound to the chromosomes. FISH is often used for
finding specific features in DNA for use in genetic counseling, medicine,
and species identification. FISH can also be used to detect and localize
specific mRNAs within tissue samples. In this context, it can help define
the spatial-temporal patterns of gene expression within cells and
tissues.

H. Ligation Probes

[0214] The advanced polynucleotide technology described herein can also be
used to practice multiplex PCR using ligation probes. Ligation probe
methods are known to those of skill in the art. Briefly, ligation probes
consist of two separate oligonucleotides, each containing a PCR primer
sequence. It is only when these two hemi probes are both hybridized to
their adjacent targets that they can be ligated. Only ligated probes will
be amplified exponentially in a PCR. The number of probe ligation
products therefore depends on the number of target sequences in the
sample.

[0215] In some embodiments, two ligation probes are separated by about 1
to about 500 nucleotides, and prior to ligation the first probe is
extended by a DNA thermostable polymerase lacking strand-displacement
activity. A DNA thermostable polymerase lacking strand-displacement
activity includes, but is not limited to, a Pfu polymerase. When the
extended strand reaches the 5'-phosphate group of the second ligation
probe, polymerization stops and a nick is created. This nick can be
sealed by a thermostable ligase present in the reaction mixture, allowing
for the entire reaction to occur in a single-reaction format.

I. Next Generation Sequencing (NGS)

[0216] The polynucleotide combinations of the present disclosure may also
be used in NGS applications. Instead of sequencing by sequential ligation
of DNA probes, the primer combinations disclosed herein can be used in
sequential hybridization without ligation. For a review of NGS
technology, see Morozova et al., Genomics 92(5): 255-64, 2008,
incorporated herein by reference in its entirety. NGS is readily
understood and practiced by those of ordinary skill in the art.

J. Insertion/Deletion (Indel) Detection

[0217] In some embodiments, and as described herein, use of a
polynucleotide primer combination of the disclosure is used to detect a
mutation in a target polynucleotide versus a non-target polynucleotide.
In various aspects, the mutation is due to a deletion and in further
aspects, the mutation is due to an insertion. Deletions and insertions
are found in the normal human population, but they are also associated
with disease and are frequently associated with human cancers.

[0218] The disclosure provides methods to detect insertions and/or
deletions in a target polynucleotide using a polynucleotide primer
combination as described herein. Quantitative PCR detection of low copy
number alleles carrying a deletion or an insertion in the presence of
high copy number wild type (i.e., normal) DNA is a comparable or more
challenging task than detection of a single base mutation (i.e.,
substitution). Use of the polynucleotide primer combinations of the
disclosure, however, affords an efficient way to detect these types of
mutations. Detection of small deletions by polynucleotide primer
combinations of the disclosure and qPCR is similar to detection of base
substitutions. The forward polynucleotide primer combination is designed
such that the approximately 6-12 base domain (f) is complementary to the
proximal and distal sequences flanking the deletion in a target
polynucleotide and where the deletion breakpoint is located in the middle
of domain (f). Domain (f) can efficiently prime and promote a
primer-extension reaction on the target polynucleotide due to formation
of an uninterrupted stable duplex between domain (f) and target
polynucleotide but cannot prime and promote a primer-extension reaction
on a non-target polynucleotide due to an unstable interrupted duplex
formed by domain (f) and the non-target polynucleotide. Specificity of
the deletion-specific primer combination to the target polynucleotide can
be further increased by adding a blocking oligonucleotide complementary
to the non-target polynucleotide that will outcompete binding by domain
(f) whose complement is not contiguous with the non-target
polynucleotide. The 3' end of the blocker polynucleotide has a group that
prevents its extension by a DNA polymerase (i.e., an extension blocker).
As a result, when combined with a reverse primer, a probe and a
thermostable polymerase, the polynucleotide primer combination can
selectively amplify and detect a small amount of DNA carrying a small
deletion in the presence of large amount of non-target DNA in a
quantitative PCR mode.

[0219] Accordingly, in various embodiments, for a relatively small
deletion of DNA in a target polynucleotide (i.e., at least 1, 2, 3, 4, 5,
6, 7, 8, 9 or 10 nucleotides) it is contemplated that the sequence of
domain (f) is identical to a mutant target polynucleotide wherein up to
about 10 nucleotides are deleted relative to the wild type (non-target)
polynucleotide (FIG. 26). In further aspects, a blocker polynucleotide is
used to reduce the priming and extension of a non-target polynucleotide
by specifically hybridizing to the non-target polynucleotide (FIG. 27).

[0220] In further embodiments, for a larger deletion of DNA in a target
polynucleotide (i.e., more than about 10 nucleotides) (FIG. 28).
Detection of larger deletions by a polynucleotide primer combination of
the disclosure and qPCR is different from detection of base substitutions
or small deletions. In the case of a large deletion, the forward
polynucleotide primer combination is designed such that the approximately
6-12 base domain (f) is complementary to both the target polynucleotide
region (F) and the non-target polynucleotide region (F*) flanking the
5'-distal breakpoint of the deletion. Domain (f) can efficiently prime
and promote a primer-extension reaction on the target polynucleotide due
to formation of a stable complex between domain (f) and the target
polynucleotide but cannot with the same efficiency prime and promote a
primer-extension reaction on a non-target polynucleotide due to the
distance between regions (A) and (F). However, priming of a non-target
polynucleotide cannot be excluded completely for relatively short
deletions (for example and without limitation, approximately 8-50 bases)
due to formation of complexes with looped-out non-target polynucleotide
templates. Such looping-out can be efficiently reduced or completely
precluded by hybridization of a blocker polynucleotide comprising the
deleted region that can extend through region (F) (see FIG. 28). The
higher rigidity of double-stranded DNA formed within the putative
deletion region will prevent loop formation, thus preventing domain (f)
binding and primer extension on a non-target polynucleotide. The 3' end
of the blocker polynucleotide has a group that prevents its extension by
a DNA polymerase (i.e., an extension blocker). As a result, when combined
with a reverse polynucleotide primer, a probe and a thermostable
polymerase, the polynucleotide primer combination can selectively amplify
and detect a small amount of DNA carrying a larger deletion in the
presence of a large amount of non-target polynucleotide in a quantitative
PCR mode. The method described above does not require a precise, base
resolution knowledge of the deletion boundaries. For this reason the
method can be used for the simultaneous (multiplexed) detection of
several potential deletions localized within a certain genomic site, each
with distinct breakpoint s. The multiplexed method may utilize one first
polynucleotide (1) or require several different first polynucleotides
(1).

[0221] In still further embodiments, a polynucleotide primer combination
of the disclosure further comprises a rigid frame blocker polynucleotide
(RF). FIG. 29 provides a depiction of a rigid frame blocker
polynucleotide. A rigid frame blocker polynucleotide is formed by
hybridization of two polynucleotides RF1 and RF2 with complementary
internal sequences (r) and (v). These sequences form an approximate 30-50
base-pair double-stranded stem, while terminal sequences q, t, u and v
remain single-stranded. Sequences q and u are designed to be
complementary to the 3' end of the deleted region ("3' DEL" in FIG. 29),
sequences t and w are complementary to the 5' end of the deleted region
("5' DEL" in FIG. 29). The 3' ends of the polynucleotides RF1 and RF2
have a group that prevents their extension by a DNA polymerase (i.e., an
extension blocker). Interaction of the rigid frame blocker
polynucleotides with wildtype (i.e., non-target) DNA (FIG. 30) results in
the formation of a "Rigid Frame" blocker-wildtype DNA complex where
regions (A) and (F) are separated by a distance sufficient to prevent
simultaneous binding of domain (a) and domain (f) of the polynucleotide
primer combination to regions (A) and (F), respectively, and extension of
domain (f) by a DNA polymerase.

[0222] In another embodiment, a polynucleotide primer combination of the
disclosure is used to detect a deletion in a target polynucleotide,
wherein the second polynucleotide (2) comprises a junction-specific
domain (f). Detection of large deletions by a polynucleotide primer
combination of the disclosure and qPCR is achieved using a forward primer
designed such that the 6-12 base priming domain (f) is complementary to
the junction region formed by DNA sequences flanking the deleted fragment
with the junction located in the middle or close to the middle of the
priming domain (f) (for example and without limitation, if domain (f) is
8 nucleotides in length, then the junction is located at base number 3,
4, 5, or 6 from the 3' end of domain (f)). Domain (f) can efficiently
prime and promote a primer-extension reaction on the mutant (deleted) DNA
due to formation of a stable complex between domain (f) and the mutant
DNA but cannot prime and promote a primer-extension reaction on wildtype
DNA due to a lack of complementarity between the wildtype DNA and 3'
portion of domain (f). Formation of a complex between domain (f) and
looped-out wildtype DNA at high temperature (approximately
50°-70° C.) is unlikely due to very low stability of a such
complex even at room temperature. Such looping-out of wildtype DNA can be
further reduced or even completely precluded by hybridization of a
blocker polynucleotide complementary to the deleted region. As a result
of higher rigidity of double-stranded DNA formed within the putative
deletion region of wildtype DNA, the loop formation will be prevented and
any potential complex between domain (f) of the polynucleotide primer
combination and wildtype DNA will not be formed and extended by DNA
polymerase. As a result, when combined with a second reverse primer, a
probe and a thermostable polymerase the polynucleotide primer combination
can selectively amplify and detect a small amount of DNA carrying a long
deletion in the presence of large amount of wildtype DNA in a
quantitative PCR mode. The method depicted in FIG. 31 requires a precise,
base resolution knowledge of the deletion boundaries. For this reason it
would require several second polynucleotides (2) with different binding
domains (f) that are complementary to corresponding deletion junction
regions if used for the simultaneous (multiplexed) detection of several
deletions localized within a certain genomic site. The multiplexed method
may utilize one first polynucleotide (1) or require several different
first polynucleotides (1).

[0223] In some embodiments, a polynucleotide primer combination of the
disclosure is used to detect an insertion of one or more nucleotides in a
target polynucleotide (FIG. 32). Detection of small insertions by a
polynucleotide primer combination of the disclosure and qPCR is similar
to detection of base substitutions and small deletions. A forward primer
is designed in such a way that the approximately 6-12 nucleotide priming
domain (f) is complementary to the mutated region and the insertion is
located in the middle of the priming domain (f). Domain (f) can
efficiently prime and promote a primer-extension reaction on the mutant
(inserted) DNA due to formation of a stable uninterrupted duplex between
domain (f) and the mutant DNA but cannot prime and promote a
primer-extension reaction on wildtype DNA due to instability of the
interrupted duplex formed by domain (f) and wildtype DNA. Specificity of
the insertion-specific polynucleotide primer combination to the mutant
template can be further increased by adding a blocker polynucleotide
complementary to the wild DNA template that will outcompete priming
domain (f) for binding to wildtype DNA (see FIG. 33). The 3' end of the
blocker polynucleotide has a group that prevents its extension by a DNA
polymerase (i.e., an extension blocker). As a result, when combined with
a reverse primer, a probe and a thermostable polymerase, the
polynucleotide primer combination can selectively amplify and detect a
small amount of DNA carrying a small insertion in the presence of large
amount of wildtype DNA in a quantitative PCR mode.

[0224] In an embodiment, a polynucleotide primer combination of the
disclosure is used to detect a large insertion (at least about 8
nucleotides in length) (FIG. 34). In some aspects, domain (f) of the
polynucleotide primer combination is designed to be complementary to the
3' end of the inserted region. Domain (f) forms a stable complex with
mutant DNA and becomes extended by a DNA polymerase, but domain (f) does
not form a complex with wildtype DNA and cannot be extended by a DNA
polymerase. As a result, when combined with a reverse primer, a probe and
a thermostable polymerase, the polynucleotide primer combination can
selectively amplify and detect a small amount of DNA carrying a large
insertion in the presence of large amount of wildtype DNA in a
quantitative PCR mode.

[0227] Other labels besides fluorescent molecules can be used, such as
chemiluminescent molecules, which will give a detectable signal or a
change in detectable signal upon hybridization, and radioactive
molecules.

[0228] In some embodiments, the second polynucleotide comprises a quencher
that attenuates the fluorescence signal of a label. In other embodiments,
the fourth polynucleotide comprises a quencher that attenuates the
fluorescence signal of a label. Quenchers contemplated for use in
practice of the methods of the disclosure include but are not limited to
Black Hole Quencher 1, Black Hole Quencher-2, Iowa Black FQ, Iowa Black
RQ, Zen quencher, a G-Base, and Dabcyl.

[0229] The references cited herein throughout, to the extent that they
provide exemplary procedural or other details supplementary to those set
forth herein, are all specifically incorporated herein by reference.

EXAMPLES

[0230] A person of skill in the art will appreciate that when primers or
primer combinations are referred to as being in "forward" or "reverse"
orientations, these designations are arbitrary conventions used in
describing polymerase chain reactions (PCR) and the structural
relationship of the primers and the template. Thus, as is apparent to a
person of skill in the art, re-orienting a PCR schematic diagram by
flipping it 180° would result in "forward" primers becoming
"reverse" primers and "reverse" primers becoming "forward" primers, and
as such, designation of, for example, one primer combination as a forward
primer or a reverse primer is not a limitation on the structure or use of
that particular primer combination.

Example 1

Real Time PCR Using Primer Combinations of the Disclosure where Region (F)
is 5' to Region (A) in the Target Polynucleotide**

[0233] Template DNA: To generate KRAS mutant DNA templates for real time
PCR, Promega WT genomic DNA was spiked with the designated amount of
synthetic KRAS G12V genomic DNA so that number of mutant KRAS copies
varied from one to 14,000 copies per 50 ng of total DNA, then template
DNA was aliquoted and stored at -20° C. until use.

[0240] Data above demonstrated sensitivity level of 0.01% or 1 mutant DNA
molecule in a context of 14,000 copies of WT genomic DNA while
maintaining absolute (zero background) selectivity for the six out of
seven mutations. Such parameters of the assay satisfied the most
demanding characteristics of a diagnostic assay including rare cancer
cell detection and noninvasive diagnostic.

[0242] Second polynucleotide (2) G12V specific: 10-256, where the G12V
specific base is at the 3' terminal position of the priming domain and
has a 1 base overlap with the WT specific blocking domain of the first
polynucleotide (1).

[0243] First polynucleotide (1) WT KRAS specific blocking domain: 10-257,
where the WT specific base is the 5' terminal position of the blocking
domain and has a 1 base overlap with the G12V specific priming domain
(f).

Real Time PCR Using Primer Combinations of the Disclosure for KRAS G12S,
G12D and G13D Mutations Wherein the Blocking Oligonucleotide not Only
Overlaps Entirely with Region F* but Also Overlaps Either Partially or
Entirely with Region A and Wherein Region b Of the First Polynucleotide
can be Absent (i.e., Comprised of 0 Bases)

[0258] Template DNA: To generate KRAS mutant DNA templates for real time
PCR, Promega WT genomic DNA was spiked with the designated amount of
synthetic KRAS G12S, G12D and G13D genomic DNA so that number of mutant
KRAS copies varied from 10 to 100 copies per 3.6 nanograms (ng) of total
DNA, then template DNA was aliquoted and stored at -20° C. until
use.

[0264] qPCR curves for 3 different KRAS mutant samples containing 10 and
100 copies of KRAS G12S, G12D and G13D in a background of 3.6 ng of
wild-type genomic DNA and a negative control curve showing background
amplification from 3.6 ng of wild-type genomic DNA.

Conclusions

[0265] Data described above demonstrated a sensitivity level of 1% or 10
mutant DNA molecules in a context of 1,000 copies of WT genomic DNA while
maintaining absolute (i.e., zero background) selectivity for the 3 KRAS
mutations. Such parameters of the assay satisfied the most demanding
characteristics of a diagnostic assay including rare cancer cell
detection and a noninvasive diagnostic.